Glycoproteins and Glycolipids in
Disease Processes
Earl F. Walborg, Jr., EDITOR The University of Texas System Cancer Center
A symposium sponsored by the ACS Division of Carbohydrate Chemistry at the 175th Meeting of the American Chemical Society, Anaheim, March 14-15, 1978.
ACS SYMPOSIUM SERIES 80
AMERICAN
CHEMICAL
SOCIETY
WASHINGTON, D. C. 1978
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Data
Library of Congress
Glycoproteins and glycolipids in disease processes. (ACS symposium series; 80 ISSN 0097-6156) Includes bibliographies and index. 1. Glycoproteins—Congresses. 2. Glycolipids—Congresses. 3. Tumor antigens—Congresses. 4. Carbohydrate metabolism disorders—Genetic aspects—Congresses. I. Walborg, Earl F. II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series : American Chemical Society. ACS symposium series; 80. QP552.G59G6 ISBN 0-8412-0452-7
616.3'99 ASCMC8
78-11920 80 1-480 1978
Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ACS Symposium Series Robert F. Gould, Editor
Advisory Board Kenneth B. Bischoff
Nina I. McClelland
Donald G. Crosby
John B. Pfeiffer
Jeremiah P. Freeman
Joseph V. Rodricks
E. Desmond Goddard
F. Sherwood Rowland
Jack Halpern
Alan C. Sartorelli
Robert A. Hofstader
Raymond B. Seymour
James P. Lodge
Roy L. Whistler
John L. Margrave
Aaron Wold
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide
a medium for publishin format of the SERIE parallels that of the continuing I N CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PREFACE T t is fitting that the proceedings of this symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the life and scientific contributions of my friend and fellow scientist, Ward W. Pigman. Much of Wards professional effort was devoted to the interchange of scientific information, both by his editorial skills and his support of national and international symposia on glycoconjugate chemistry. This symposium was born in Ward's mind in 1976 during deliberations of the Division of Carbohydrat Society concerning symposia topics for the coming years. Ward felt that sufficient progress had been made concerning the relationship of glycoconjugates to a variety of disease processes to warrant a symposium devoted to this topic. At that early date, the Carbohydrate Chemistry Division made a commitment to sponsor a symposium on some aspect of glycoprotein research and designated Ward as chairman with responsibility to formulate the program. Early in 1977, Ward informed me of his plans for the symposium and asked if I would serve as co-chairman. I gladly agreed and we set about the task of preparing a tentative program and contacting prospective speakers. On September 28, 1977, during the Fourth International Symposium on Glycoconjugates being held at Woods Hole, Ward and I finalized the symposium program. Upon completion of this task Ward expressed to me his personal satisfaction that the years of basic research on glycoconjugates wasfindingincreasing relevance to understanding a variety of disease processes. Ward's sudden death on September 30, 1977, was a shock to the many participants of the Wood's Hole meeting, many of whom were his long-time friends. In a way it was fitting that his last days found him engaged in an international meeting on glycoconjugates, an endeavor he had vigorously promoted and fostered for many years. Many of the participants in the symposium on "Glycoproteins and Glycolipids in Disease Processes" were present at the Wood's Hole meeting and by common consent it was decided that the planned symposium and this monograph should be dedicated to Ward Pigman's life and contributions to glycoconjugate research. The efforts of many persons and agencies were responsible for the success of the symposium and the publication of this monograph. First, I should like to express my appreciation to all of the participants for their contributions to the symposium and to the monograph. The supix In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
port of the Division of Carbohydrate Chemistry, particularly the efforts of Derek H. Ball and Gary D. McGinnis, is gratefully acknowledged. Grants from the National Institute of General Medical Sciences (GM 25226) and the National Cancer Institute (CA 24513), which provided travel support for participants in the symposium, are gratefully acknowl edged. Particular gratitude is extended to Edward Hampp (N.I.G.M.S.) and Mary A. Fink (N.C.I.) for their efforts in making this symposium a reality. On numerous occasions I have been encouraged by Gladys Pigman. Able assistance was obtained from Beverly Sea, Carey Osborn, Reita Maughan, and Pat McKirahan, staff at the Research Division— Science Park, The University of Texas System Cancer Center. Science Park—Research Division
E A R L F. WALBORG JR
The University of Texas July 20, 1978
χ
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
To Ward W . Pigman
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
DEDICATION TO WARD W. PIGMAN It is appropriate that this Symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the memory of my friend and colleague, Ward W. Pigman, who passed away on September 30, 1977, during his attendance at the 4th International Meeting on Glycoconjugates being held at Woods Hole, Massachusetts. Ward convinced the Carbohydrate Division of the American Chemical Society that a symposium on this subject warranted consideration for the spring meeting of the ACS to b efforts and those of Earl F. Walborg, Jr., this symposium has become a reality. Barely two hours before Ward's death, I had the opportunity to discuss his plans for this symposium, and I realized how deeply he felt about it. In a way, this symposium represents the ultimate goal of the long scientific road he and quite a few others among us have traveled, a road through carbohydrate chemistry that has led us from the use of analytical and physical chemistry in the study of reaction mechanisms to synthetic chemistry andfinallyto the elucidation of the structure of more and more complex carbohydrates, including glycosaminoglycans of connective tissue and glycoproteins from mucous secretions and the surface of the cancer cell. I feel that I have a deep understanding of Ward's research interests because both of us followed quite similar pathways, becoming interested in connective tissue polysaccharides in the late forties. In 1930 at the age of 20, he joined the National Bureau of Standards, where he was active until 1943. During this time, he obtained his B.A. and MA. degrees at George Washington University and his Ph.D. degree at the University of Maryland. At the Bureau he was associated with Horace Isbell, one of the leading American carbohydrate chemists. A series of papers reflecting Isbell's interests in the physical and organic chemistry of carbohydrates resulted from this collaboration. Soon a change in Ward's interests was noticeable, a turn to more biological problems with a study of glycosidases. This turning point was marked by a paper published in collaboration with H. Isbell and B. Helferich and by Ward's obtaining a Lalor Fellowship to spend one year in Leipzig, a stay that was unfortunately interrupted by the start of World War II. He returned to the National Bureau of Standards, and then during the last two years of the war he became a group leader at Corn Products, where most of his efforts were directed toward starch chemistry. Following his brief assoxiii In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ciation with Corn Products, he moved to the Institute for Paper Chemistry at Appleton, Wisconsin, an institute for basic research supported by the industry, where he also served as a group leader. During the Wisconsin period, Ward's interests included pectins, cellulose, carbohydrate-degrading enzymes, and tannins. In 1949 Ward moved into the academic community, accepting a position at the University of Alabama Dental School where he was Associate Professor until 1960, Professor and then Dean of the Graduate School from 1963 to 1968. He remained associated with this institution as a Visiting Professor until his death. In 1960 Ward assumed the additional burden of a second academic position as Professor and Chairman of the Department of Biological Chemistry at New York Medical College. These moves to academic positions in a dental and medical school correspond to a final shift in series of papers on the reaction between amino acids and carbohydrates, the so-called Browning reaction, initiated at Appleton, papers appeared on dental plaque and saliva, and then the beginning of the work for which Ward is now best known:first,investigations on hyaluronic acid degradation and then an outstanding series of papers on submaxillary mucins. Before this work had been started, the contribution of Ward to carbohydrate chemistry had already been recognized by his selection as the recipient of the Hudson Award by the Carbohydrate Chemistry Division of the American Chemical Society in 1959. Other awards followed, such as medals of the Société de Biochimie de France and of the University of Milan. Enumerated below are a few of Ward's contributions to glycoconjugate chemistry. (a) Elucidation of the mechanism of degradation of hyaluronic acid by ascorbic acid and free radicals. (b) Purification of submaxillary glycoproteins and elucidation of many aspects of their peptide and saccharide structure. (c) Elucidation of the ^-elimination mechanism for cleavage of the glycopeptide bond between 2-acetamido-2-deoxy-D-galactose and serine or threonine, the predominant glycopeptide linkage of the mucin glycoproteins. (d) Suggestion that the peptide moiety of the mucin glycoproteins is composed of repeating sequences of 28 amino acids. Although present evidence suggests a more complex arrangement, this hypothesis will continue to deserve attention.
xiv In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
This symposium and the resulting monograph bear the mark and vision of Ward W. Pigman, and therefore are fitting memorials to his life and scientific contributions to glycoconjugate chemistry. Harvard Medical School
and Massachusetts General Hospital Boston, Massachusetts August 21, 1978
ROGER JEANLOZ
XV In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Introduction ROGER W. JEANLOZ Laboratory for Carbohydrate Research, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114
To some chemist member Chemistry of the American Chemical Society, which is organizing this symposium, it may be unexpected that, for the next few days, we w i l l be discussing compounds that encompass lipids and proteins, in addition to carbohydrates, and their relation to disease processes. It is within the life span of many of us that natural product chemists have realized that nature is not compartmentalized into such nice and clean-cut categories as carbohydrates, proteins, and l i p i d s ; in fact, very few carbohydrates exist that are devoid of a peptide or l i p i d component, as well as there are few proteins devoid of a carbohydrate component. The roles of the carbohydrate component, of glycoproteins and glycolipids that are located at the surface of the animal c e l l or present in the connective tissue that links the c e l l s , have received increased recognition in the past decade and w i l l be discussed during the next few days. The subjects of the f i r s t session of this symposium w i l l be the methods of structure identification presently being developed, as well as the biosynthesis and degradation of glycoproteins and glycolipids. Because glycoproteins and glycolipids of biological interest are generally available only in minute amounts, the methods of structure identification, which w i l l be discussed by Dr. Walborg in his general presentation of current concepts of glycoprotein structure, are being developed at the micro- and even nano-gram l e v e l . Dr. Schachter and associates w i l l entertain us with the very active development of glycoprotein biosynthesis presently taking place. For many years, the concept of glycoprotein and glycolipid biosynthesis was dominated by the concept of elongation of the chains by one carbohydrate unit at a time, through sugar nucleotides; this concept, f i r s t developed for such homoglycans as glycogen and starch, was very ably demonstrated for glycoproteins and glycolipids by Roseman's group. Nearly twenty years ago, this unified concept of chain elongation was shown to be invalid for bacterial polysaccharides, and the importance of isoprenoid 3 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
sugar
phosphate
recognized. work
on
sugar have in
sugar
phosphates
the
intermediates
years in
the
D-glucose
etc.
It
with
the
glycoprotein
is
of
of
more
the
than
polysaccharide group,
established
in
the
the
role
role
component
the
passing
of of
transfer interest
processes:
One
^-glycoproteins
and
involves
rather
where
the
carbohydrate
takes
place
through
transfer at
l i p i d
elongation, are
The which
of
their
these
be
to
units
very
simple
components
two
the we
the
are
faced
processes
animal
and
mechanism, backbone
synthesized
degraded
coexist
now
in
peptide
before
where
sugar
the
chain,
complex
process
from
years
peptide
active
to
few
intermediates
^-glycoproteins,
oligosaccharides
and p a r t i a l l y
a
past
new
sugar
units
nucleotides.
or
are
Time
merely
the
of
blood-group
those
structures
are
found
length
at
the
to
of
eukaryotic
glycoglycerolipids
their
chain
as
b
solely
variation or
large
restricted
composition
process
is
single
residues
their
complex
in
w i l l
diversity of
of
its
inadequat
encompass
pyranosyl and
as
glycolipids
associates The
second
show w h e t h e r
expression
stage
intermediates
the
transferred
w i l l
of
the
a
is
was
for
isoprenoid
The
that
two
plant
of
known
these
to
of
synthesis
already
biosynthesis.
of
core
residues
existence
in
L e l o i r ' s
development
biosynthesis
of
ago,
nucleotides,
witnessed
role
as
Ten
the
up
to
surface
typing,
of
have
for
50
of
the
a-D-galacto-
chain
of
carbohydrate
units
the
organisms,
glycosphingolipids.
example
within
(evidence
having
result
(for
only
and
has
cells
been
of
reported),
during
the
specific
genetic
defects,
a l l
reasons
for
been
glycolipids), chains
oncogenic their
active
role
investiga
tion. Finally, the
most
microgram
specific chains
the
by
ago,
Dr.
of
we
by
were
Maley
Eylar of
D.
not
Dr.
degradation
methods the
identification of
this
his
been
are
we
and to
of
It
able
the the
more
progresses
glycoproteins these
to
my
the
I
laboratory structure such
that
present
addition of established and
structures
of
α -acid an
glycolipids disease
endo-
found
extract
that
biochemical
chemical i n
glyco
x
of
the
25
exo-
enzyme, in
be
when,
with
lack
such
recently
with
carbohydrate
remember
time
made
off
at
the
and which w i l l
the
was
elucidations
without
s p l i t
linkage
deploring
group,
is
the
in
on
were
his
structure possible
associates.
working
pneumoniae
great
correlation of
subject
was
manipulating.
allowed
chemical
have
that
r e a l i z i n g at
Kobata
has the
not
and
human p l a s m a ,
glycosidase later
recent
carbohydrate-peptide
Dr.
glycosidases protein
the would
endo-glycosidases
near
discussed years
of
level
methods
that
structural
and
made
possible
processes,
Symposium.
RECEIVED August 2, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
the
2 Biosynthesis and Catabolism of Glycoproteins HARRY SCHACHTER, SAROJA NARASIMHAN, and JAMES R. WILSON Research Institute, Hospital for Sick Children, Toronto, Canada
Glycoproteins are biosynthesis is, not unexpectedly Although much has been learned about this process in the past ten years, there are many aspects which we do not understand. In the limited space permitted for this article, it will be possible only to outline the major steps in the biosynthesis of some of the gly coprotein types; more detailed reviews are available (Schachter and Rodén, 1973; Montreuil, 1975; Schachter, 1974a,b, 1977, 1978; Waechter and Lennarz, 1976). I. Initiation of oligosaccharides of the asparagine-N-acetyl-Dglucosamine linkage type. The largest group of animal glycoproteins are those which contain only or predominantly oligosaccharides that are linked to the polypeptide back-bone by an N-glycosidic linkage between N-acetyl-D-glucosamine (GlcNAc) and asparagine (Asn). Many secreted glycoproteins (α1-acid glycoprotein and other plasma globulins, immunoglobulins, various gonadotrophins, ovalbumin, various enzymes, etc.) and membrane-bound glycoproteins (red cell membrane glycophorin, rhodopsin, the envelope glycoproteins of certain viruses such as vesicular stomatitis virus and Sindbis virus) contain Asn-GlcNAc linkage type oligosaccharides. It has become clear that the synthesis of the sugar-amino acid linkage pre-determines the general nature of the oligosaccharide that is subsequently assembled, i.e., that the synthesis of this linkage is an important control point. It is therefore important to understand the initiation process. In recent years it has become apparent that many (but not all) Asn-GlcNAc oligosaccharides share a common "core" structure (Montreuil, 1975) : Man-α1,3 Man-al,6
Man-31,4-GlcNAc-31,4-GlcNAc-Asn
0-8412-0452-7/78/47-080-021$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Further,
the
Hemming,
Heath,
1976,
and
strong
work
is
evidence
in
Soon
after
suggested cells. that
It
tissues
of
of
mammalian
core
is
scheme
Jeanloz,
and
has
Lennarz,
provided
pre-assembled prior
for
role
of
the
to
as
a
incorporation
initiation
p o l y p r e n o l - l i n k e d sugars
polysaccharide
a
process
similar however,
the
1);
be
pioneering
evidence and
nucleotides
(Fig.
might
assembly,
reaction
the
to
work
for
the
of
had
in
Leloir s
group
1
transfer
i n
mammalian
N-acetylglucosamine
from a
phosphate,
phosphate
workers
higher
dolichol
dolichol
been
various
occuring
their
phosphoryla-
sugars
formed
are,
Man-3-monophos-
GlcNAc-a-pyro-phosphate-dolichol, Glc-3
and
intermediates tissues
thyroid,
hen
ha
(rat
and p i g
oviduct,
l i v e r ,
human
mouse
myeloma
lymphocytes,
calf
tumor,
pancreas,
. Another
covery that
of
rat
glucose
liver
to
thought
to
water,
in
but
the
be
protein.
This
of
study
in
this of
the
product
product and
an
which
was
in
to
dolichol
the
was
of
radioactive
was
at
f i r s t
subsequently
shown
w h i c h was
et
aJU ,
pyrophosphate
ac to
insoluble
chloroform-methanol
(Behrens
dis
noted
endogenous
chloroform-methanol-water
solvent
was It
transfer
glucose
oligosaccharide acid
group
1
effect
acid-insoluble
soluble
The
length
of
the
tissue
and w i t h
reports
from
contain
about
mannose
and
mouse
20
residues
η
assembly
esting
to
formation
the
led
be
in
(2:1,
(10:10:3,
1971)
v/v)
the
oligosaccharides
units
residues. for
oligosaccharide of
GlcNAc
and
attached
conditions
laboratory
microsomes,
;
way in
5-7
used
indicated
to
for
the of
With
tissues
other
appeared
the to
residues
of
varies The
oligosaccharide
consisting example),
dolichol
synthesis.
to
N-acetylglucosamine, (hen
oviduct
predominant
lack
glucose
mannose,
and
as
and
dolichol contain
follows:
-Man-31,4-GlcNAc-31,4-GlcNAc-pyrophosphate-dolichol of
note of
oligosaccharide
glycose
glucose
myeloma
(Man-α-)
the
Leloir's
pyrophosphate
from
could
L e l o i r s
oligosaccharide.
tissues.
with
The
an
from
monophosphate
trichloroacetic was
intense
two
microsomes
pyrophosphate
discovery
many
contribution pyrophosphate
dolichol
form
dolichol
v/v)
major
dolichol
from
ceptor
to
reviews)
intermediate
general
mannose
l i p i d
(Leloir,
Waechter
1.
the
sugar
phate-dolichol
a
The
see
recent
common
glucose,
polyprenol
etc)
this
strong
respectively,
bovine
for
provided
of
laboratories others,
bacterial
was,
respective ted
in
that
first
1978,
that
Figure
established
and
oligosaccharide
polypeptide.
shown
several
Schachter,
lipid-linked into
of
Lennarz
this the
structure
inversions
3-linked
mannose
is of
shown
in
anomeric
from
Fig.
1.
It
is
configuration
GDP-a-Man and
inter
in
α-linked
the mannose
Man-3-monophosphate-dolichol. M.J.
carried
Spiro
out
containing
et
careful dolichol
a l .
(1976a,b)
and
investigations pyrophosphate
on
R.G. the
Spiro
et
formation
oligosaccharides
al of by
(1976)
have
glucoseseveral
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
tissues.
Working
with
these
workers
phate
oligosaccharide
Biosynthesis
thyroid
demonstrated with
slices
and
rather
than a
23
Catabolism with
the
formation
of
the
tentative
structure:
microsomes,
dolichol
pyrophos
(Man-α-) -(Glc) _ ~(Man-α-) -Man-GlcNAc-GlcNAcβ
1
2
4
PY r o p h o Evidence and
was a l s o
thymus
pyrophosphate preparations these
large
molecules lack
slices calf
dolichol
which
Parodi
for the of
pyrophosphate only
However, et
It
of
dolichol
pyrophosphat
glucose
believed
to
is
so
that
dolichol The
final
asparagine work and the to
i n i t i a t i o n process
and Lennarz, labelled
that
membrane the
that
that
is
of the
unglycosylated presence
of
by
retory
The
is
It
always
of
attached
pathway
transfer
However
to
the presence
pyrophosphate
of
of
of
further
forms
of
pathway. for
from
that
any amino sequence
the
dolichol
three
which is
sec
under
oligosaccharide
acid
does
A. effects
not
i n the tripeptide
almost this
time
i n
sec
and ribonuclease
polypeptide
some
a
glycoprotein
the transferase
for
rates
evidence
oligosaccharide
into
synthesis
ovalbumin,
the dolichol
denatured
an asparagine X can be
that
i n secretory
α-lactalbumin
recognized to
by
obtained
specificity
(Thr)-where
dolichol
has
tunicamycin,
normal
oligosaccharide
has been
i n h i b i t e d by
almost
appears
synthesis
however,
continued
oligosaccharide
substrate
Asn-X-Ser 1974).
the
suggested
since
i t
to
proteins
i n the
work,
endo
bound
secretory
only
and
The
1977);
at
been
asparagine
i n i t i a l l y
of
cells
intermediate
a l l tightly
recent is
l i p i d
an
catalyzed
reactions.
known
synthesis
have
the d o l i c h o l
showing
More
myeloma
has not yet
between
involved
glycosylated
proteins-ovalbumin,
incorporation stood.
is
(1977)
i t
of
to
Earlier
and Lennarz,
tunicamycin,
glycoprotein,
synthesis
the
from
findings
be
transfer
preparations
were as
ovalbumin
ovalbumin
and Lennarz
pyrophosphate
of
(Struck
retory
of
these
might
the
(Fig. 1).
i n these
identified
blocks
Pless
involvement
linkage
glycoproteins.
which
glucose-
configuration.
is
rat l i v e r ,
however,
experiments
study;
glycosylation
antibiotic
with
microsome
synthesized
pathway
N-acetylglucosamine
and
et a l . ,
oligosaccharide
chain
oligosaccharide
n o t be
under
dolichol
membrane-bound
shown an
tissues
the
1976) that
acceptors;
i n these
and could
smaller
(Herscovics
large
anomeric
pyrophosphate
an N - g l y c o s i d i c
acceptors
of
i n the polypeptide
N-acetylglucosamine
of
dolichol
protein
but
glucose-3-monophosphate-
from
i n the
of
microsome
produce
(Behrens
pancreas
step
transfer
genous
calf
synthesizing
glucose
established
established
of
of
hen oviduct endogenous
rat liver
inversion
residue
(Waechter
from
single
from
that
do n o t
and N-acetylglucosamine
a
oligosaccharide
kidney dolichol
b
transfer
involves
interesting
oligosaccharides
and from
capable
containing cules
is
mannose
microsomes
a l . , 1972) were
by o v i d u c t ,
glucose-containing
t h y r o i d and hen oviduct
contain
a l . , 1977a,b)
formation
similar
oligosaccharides. from
glucose.
1971; et
obtained
tissue
sphate-do1icho1
sequence(Marshall,
not
necessarily
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
result a
i n
glycosylation
protein
may,
cosylated (Thr)The
forms
site
work
but
that
A l l
may
be
completely
lactalbumin sequence
the
three and
ineffective
fully
protein A)
must
the
required tripeptide
and
the
Although serve
smaller This
into
observation transfer that
GlcNAc
contain
structures clear by
such
and
oligosaccha-
have
not
as yet
which
lack
and
since
1975)
o fa l l
glucose
stomatitis stages in
1978).
involve The
t othis
the
the
The
viral
class
Work
and
Sindbis
assembly
leads
e ta l . , envelope
plasmacytoma processing
Asn-GlcNAc
oligosaccharides
oligosaccharide i n
2.
F i g . may
f o l -
mannose
evidence has
i n d i -
envelope
subsequent 1977;
glyco-
rough
processing
o f mannose Robbins,
glycoproteins processing since
c e l l
(Tabas
line
GlcNAc,
Man a n d
endoplasmic
are
reprehowever,
biosynthesis o f was
e tal_.,
initiation
(and
1977;
i s , also
shown
1978) .
and
I t i spostulated
undergo
containing the
be
enveloped
virus)
t o removal
glycoproteins;
in
containing
must
the
o f the
type
type
residues, i t
reported with
o fAsn-GlcNAc oligosaccharides
summarized
occurs
Asn-
a n d o f some
recently
o fglycoprotein,
a mouse
are
process
o f the
o f mannose
residues;
(Hunt
processing large
large
N-
protein-bound oligosaccharide i s
mannose
oligosaccharide
initiation
have
virus
in
viruses,
rich
by
the
contain
3 mannose
residues
processing".
o fmembrane-bound IgG
oligosaccharides. primarily
oligosaccharide
the
residues
confined
oligo-
the
Asn-GlcNAc linkage
only
11 r e s i d u e s
glucose)
secretory
i n
than
N-acetyllactosamine
and
laboratories
a tearly
e ta l . ,
effective
acceptors
which
o f the the
contain
probably sentative
of
type.
o f glucose
and
more
involves
incorporation o fa large
o f these
moieties
glucose.
glycoproteins
This
accept
incorporation
pyrophosphate
envelope
Tabas
the
protein
pathway
these
a
not
were
proteins
oligosaccharide
9 times
oligosaccharides
(vesicular
large
i n
oligosaccharides
glucose
Several
that
proteins quite
vivo
"oligosaccharide
viruses
to
denatured
o fp r o t e i n - b o u n d o l i g o s a c c h a r i d e s
removal
residues.
cated
dolichol
(Montreuil,
that
residues
lowed
a
tripeptide
sequence
oligosaccharides
small
donors
about
mannose
linkage Since
not
are
pyrophosphate
Processing
not
glycosylation t o
tha
i n
acetylglucosamine, II.
relatively
t o endogenous
glucose-free
suggests
dolichol
of
site.
protein
oligosaccharides
saccharide
for
this
did
such
acceptors
(ovalbumin, a -
glycosylation
pyrophosphate
carry
asoligosaccharide
important phate
1-2
for
for
ungly-
-Asn-X-Ser
exogenous
several
and
a tanother
required
without
sequence
requirements
dolichol
and which
oligosaccharide
is
a tone
proteins
1977);
Lennarz,
resolved.
glucose
do
with
the
and
glycosylated
unfolded
however,
ride
can
be
acceptor
with been
(1977)
proteins
asacceptors;
Pless
both
glycosylated
contained
several
exact
i n
unglycosylated
Lennarz
effective
RNase
whereas
be
exist
or
and
1974;
(Marshall,
example,
o fPless
indicates occur.
for
by Glc
reticulum
subsequent that
a l l
transfer (Fig. (see
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
of
2). next
2.
Glycoprotein
SCHACHTER ET AL.
Biosynthesis
25
and Catabolism
UOP-GicNAc Dd-P *UMP G l c N Acc*-PP- - P - D o l U D P ^ G l c N A c J-
(GOP^Mon) (GOP^M •
n 1
n
γυΟΡ
(Dol-P)4
(
n
GlcNAc GlcNAc*P-P-Dol IcNAc^P-P-Dol GDP-Man *GDP
ί I " ^Man^P-Dol)
£
Mon-GlcNAc^GlcNAc^P-P-Dol—^(Man) *Man*GlcNAc*GlcNAc*P-P-Dol n
"
l U 0 1
P
Acceptor
)
Nooi-p-p ( Man\,
Man - GlcN Ac - GlcN Ac * A S N
Figure 1. Initiation of Asn-GlcNAc-type gosaccharides are preassembled tions: Dol,
oligosaccharides.
Oli
Peptide ^
(Glc) -(Man) -(GlcNAc) -P-P-Dolichol 2
n
(Glc) -(Man) -(GlcNAc) -Asn2
2
n
2
Oligosaccharide processing
V M-
Gn-Tr I
Gn-3l,2-M-cyl,3\ Μ-β1,4-Gn-Gn-Asn-
<
(MM)
(MGn) Gn-M^ M-Gn-Gn-AsnGn-pl,2-M-cyl,3
N
M-Gn-Gn-AsnGn-pl,2-M-ol,6
x
(GnGn)| Gn-My M-Gn-Gn-AsnGn-M^ Fuc
V
α
à2,6 G G βΙΛ pl,4 Gn Gn Pl,2 pi,2 M M 1,6\ /ol,3 M
6n
G-pl,4-Gn-M
Gn-pl,2-M-cyl,3, G-pl,4-Gn-M
x
1
/
Gn-(Fuc) -Âsnn-Çn-As (Fuc)
(GS)
G - p l , 4 - G n - p l , 2^-0*1,6' (GG) (GGn)
Figure 2. Biosynthesis of Asn-GlcNAc oligosaccharides of the N-acetyllactosamine type. Initial transfer of oligosaccharide to peptide can involve a glucose-containing intermediate. Processing to the Man GlcNAc Asn core (MM) is then believed to occur, followed by the Golgi-located elongation reactions. 3
Abbreviations:
M,
Ό-mannose;
2
Gn,
N-acetyl-O-glucosamine;
Asn,
asparagine;
Fuc,
-L-fucosc;
G,
Ό-gahctose; S, sialic acid; Tr, transferase. The glycopeptides are named according to the sugars present at the nonreducing ends; the sugar on the Man-al,6-branch is named first.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
26
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
section). type of
Oligosaccharides
(Montreuil,
glucose
1975)
residues destined
(Montreuil,
1975)
residues
t o become
undergo
Man3GlcNAc2Asn
undergoes
elongation
acid,
section
not
noside
type
while
interesting primitive
others
suggestion
the processing structures
Subcellular Fig.
membrane-bound for
secretory
these
to
site
3
of
when
now c o n s i d e r a b l e
secretory
the
nascent
Blobel
peptide
across
this
theory
nascent
peptide
binding
of
reticulum nascent
a
peptide
to
a
a
r
the more
e
of
oligo-
biogenesis
has been 1977)
proposed
site
creates
a
membrane.
moieties
Toneguzzo
the
which
"signal
the
causes
endoplasmic f o r passage
of
of
hypo-
end o f
somehow
on the
channel
and
membrane,
terminal
the i n t r a v e s i c u l a r space
of
the transfer
reticulum
sequence"
that
but are
ribosomes
To e x p l a i n
t h e amino
a
except
the plasma
the peptide
of
suggested
membrane
a l . , 1977;
receptor
and thereby
into
with
have
"signal
to the
structures.
of
to
the endoplasmic that
i s
organisms
conversion
1974a,b,
that et
made
structures
type
path
1978).
(1975a,b)
the ribosome ( F i g . 3)
fuse
Wirth
proposes
carries
have
on membrane-bound
1974a,b,
It
the oligoman-
extensively
scheme
attached
evidence
core
initiation.
similar
vesicles
the then
apparatus.
higher
enable
(Schachter
1975;
Schachter
and Dobberstein
thesis" ;
a
are translated
and Lodish,
1975;
to
Asn-GlcNAc
do n o t r e m a i n
secreted
(Morrison
more
and that
of a l l
form
become
(1978)
the subcellular
There
Ghosh,
scale
apparatus
glycoproteins
glycoproteins
et. a l .
to
F u c , G a l and s i a l i c
the oligomannoside
glycoprotein;
glycoproteins i s
are processed Tabas
type
(removal
Golgi
N-acetyllactosamine
shows
Oligo-
structure
GlcNAc,
oligosaccharides
that
developed
core
i n the c e l l ' s
i n the evolutionary
mannoside I l l .
This
(loss
residues).
residues)
(the a d d i t i o n o f
structure.
3
mannose
processing
(Fig. 2).
why some
oligomannoside
the N-acetyllactosamine
extensive
IV below)
yet understood
Man GlcNAc2Asn
some
the
limited processing
a n d a l l b u t 3 mannose
structure see
t o become
undergo
and possibly
saccharides glucose
destined
probably
the
of
endoplasmic
reticulum. It
has been
shown
for several
acetylglucosamine
i s
s t i l l
the ribosome
attached
Molnar
and Sy,
1970). be
to
If
Sherr
the dolichol
concluded
peptide.
1967;
incorporated
that
Kiely
et
a l .
a n d mannose
were
bound
to
ribosomes.
It
ments
whether
peptide of has
stage
peptide been
poration
o r whether
from
of
should
have
present
while
Jamieson
Cowan
has
shown these
takes
also
that
that
(1977)
i t can nascent
both
glu-
chains
various
place
occurs
found
i s
into
ovalbumin
on the nascent
and Lodish
1,2),
incorporated
from
core
i t
and Robinson,
(Figs.
at
experi-
the
after
C o n f l i c t i n g evidence (1977)
occurs
Rothman
1966;
on nascent
process
the ribosome.
carbohydrate
while
i n fact
was n o t c l e a r this
be
some N -
and Schachter,
operative
also
a l l incorporation of
obtained;
glycoprotein
is
that
peptide
and U h r , 1969;
(1976)
cosamine
tissues
nascent
(Lawford
pathway
mannose
into
on this
l i t t l e
peptide
suggest
nascent
release point
incor-
of
that
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
oxacid the
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and Catabolism
UOS R I B O S O M A L S U B U N I T mRNA
60S
RIBOSOMAL
SUBUNIT
HYDROPHOBIC SEQUENCE
- mRNA
CYTOPLASMIC ^ F A C E LIPID B I L A Y E R O F R O U G H ENDOPLASMIC
RETICULUM
TTTTTTTTT Hiiiliii
ÎÎÎÎÎÎÎÎ--" UUUU- -" * \
RIBOSOME BINDING PROTEIN '
INTRAVESICULAR FACE
mRNA
GLYCOSYLTRANSFERASE
EXTRACELLULAR INTRACELLULAR _
FACE
FACE
Figure 3. A scheme depicting biosynthesis of a membrane-bound glycoprotein. Translation probably starts on a free ribosome and the nascent peptide carries some sort of "signal" near its amino-terminal end. This signal organizes the appearance of a ribosome-binding protein in the endoplasmic reticulum and thereby creates a channel for passage of nascent peptide through the membrane. Oligosaccharide is incorporated into peptide within the channels of the rough endophsmic reticulum (stages 5 and 6). The signal sequence is probably cleaved off at some stage in assembly although there can be exceptions. The glycoprotein migrates to the Golgi apparatus for the elongation reactions (stage 7) and then to the plasma membrane (stages 8 and 9). An analogous scheme can be drawn for secretory glycoproteins except that the glycoprotein would not be firmly anchored to the membrane during the assembly process.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
27
28
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
entire
oligosaccharide
during
assembly
t i t i s
virus.
that,
i n their
through ation; and
of
In
fact,
both
processes
the ribosome.
data
of
Jamieson
out
before
report
involved phate
worked
endoplasmic vitro
as
(Vargas
reticulum.
a l .
these
peptide
(1978)
at
locus
liver. The
shown
different;
they
contained
mole,
levels
process
this
mannose
for
7
some
type.
not
i n
nascent
to
of
Thus,
of
compatible
acid
of
with
containing mannose.
oligosaccharide endoplasmic
oligosaccharide
the low
plasma
plasma
and the
a n d showed
to
t h e ZZ that
but
of
per
2 residues
residues In
into
individuals
ZZ l i v e r s
completion.
of
suggest
incorporated normal
i t
carried
levels
strongly
mannose
whereas
of
isolated
or galactose
normally
has
significantly
(1978)
reticulum; of
by the
acid)
protein
not
results
ZZ
1978).
on the average
i s
from
ejt a ^ . ,
and increased
These
by removal
the protein
liver
a^-antitrypsin
3 oligosaccharides
per oligosaccharide)
process
of
of
i n
and lung
i n secretion
liver
II
(protease
oligosaccharides
ejt a l .
s i a l i c
kidney
(Jeppsson
human
i n section
for glutamic
3-4
i s
subsequently
a-^-antitrypsin
studies
however,
carry
Hercz
by
interesting
t h e P-^
l i v e r ,
the normal were,
an
and
at
retention
N-acetylglucosamine
i n the rough
reason,
of
specimen
glucose,
residues
residues
to
microscopic
plasma
appeared
were
a mannose-rich
polypeptide 3
of
low l e v e l s
are prone
i n t h e ZZ p r o t e i n
oligosaccharide
and
f i r s t
similar
described
(lysine
an autopsy
the data
each
GlcNAc that
both
as
substitution
from
no f u c o s e ,
decreased mannose;
the
glycoprotein
reticulum
a blockage
N-acetyllactosamine from
rough
deficiency
secretory
substantial
compositions
isolated
protein
i n a
provided
a r e due t o
reported
carbohydrate
a
have
reticulum vesicles.
acid
ZZ p r o t e i n the
a
protein
An amino been
enzymes pyrophos
i n the
however,
for the Ζ allele
relatively
and electron
endoplasmic
recently
were,
processing"
individuals
have
this
l i v e r ,
homozygous have
such
Light
of
the
providing
acceptors
endoplasmic
"oligosaccharide
rough
latter
carried
dolichol
enriched
Institute
i n human
Persons
in
the
be
preliminary
that
hen oviduct,
acceptors
this
(above) .
levels
A recent
the
are not
1977) .
undergoes
individuals
peptide
while
must
c a r r i e d out on α^-antitrypsin
i n the rough
disease.
protein
from
of
between
(1977)
studies
glycosyl
Ra
that,
sera;
were
from
to
insertion
release
indicated
oligosaccharide
confirmation
inhibitor)
secretory
oligosaccharide
glycosylated
their
a
to protein
and Carminatti,
et
coupled
membrane
before
evidence
peptide
f o r the
Studies Hercz
is
stage stoma
strong
nascent
and Lodish
(1977)
prepared
endogenous
system;
of
peptide
vesicular
for the discrepancy
c a n be made.
and Lennarz
fraction
well
of
provide
without
but further
generalizations
evidence
served
with
protein
oligosaccharide
direct
not occur
a n d Rothman
i n transferring
microsome
workers
r e t i c u l u m membrane
does
(1977)
by C z i c h i
the nascent
insertion
The reasons
a membrane
at
glycoprotein
are completed
the former
studied
added
system,
the endoplasmic
from
i s
the latter
i n vitro
glycosylation
known;
core
the envelope
(to cannot,
normal
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
livers,
the protein
(addition below;
added
normally,
that
interferes
out
of
where
the
complete
the
rough
since
with
some
apparatus
for
elongation
residues,
see
section
contain
is
IV.
Elongation within
which
reticulum to
The blockage
a
the
protein
the
pathway
sugars
must
a^-antitryp-
of
processed,
external
liver
subsequent
of
region
i n this is
and i t
or
movement
ZZ
processed
i n ZZ
glycosylation
IV
is
i n the
are
not defective
ZZ a ^ - a n t i t r y p s i n
the
fucose
The d e f e c t
or prevents
for addition of
into
c e l l
i s
not
moves
and i s
to
the
secreted
plasma.
oligosaccharides As curtail
apparatus
acid
substitution
normal
endoplasmic occurs.
acid
29
Catabolism
glycoproteins
apparatus
amino
and
Golgi
apparatus). plasma
processing,
processing
Golgi
Golgi other
either
oligosaccharide
the
n o r ZZ p r o t e i n s
the processing
postulated
sin
normal since
to
Gal and s i a l i c
i n the
n o t known;
be
travels
GlcNAc,
neither
usually is
of
Biosynthesis
pointed
processing
oligomannoside will
not be
fate
of
the
out of
type
Golgi
apparatus
of
Asn-GlcNAc
o i n section
II
(above),
oligosaccharides
are n o t known;
considered
further.
oligosaccharides
which
this
In
the
destined type
this
of
which
become
the
oligosaccharide
section,
are processed
factors
to
to
we d e a l
the
with
the
core
structure : Man-al,3 Man-31,4-GlcNAc-31,4-GlcNAc-Asn
^ Man-al,6 Fig.
2
shows
process acid
subsequent addition
i n the
end o f
Golgi
this
The fied
p r i m a r i l y by
the
toxic
proteins has tain
et
on the
at
least
the use o f
of c e l l
the
structure II.
i n wild
be
of
G a l ,
these
discussed
a
sugar,
a
GlcNAc
f o r nomenclature)
This
s i a l i c
sugars
briefly
are at
residue,
has been
I,
Evidence type
cells
(Stanley c e l l
ejt a l . ,
line
because
was
a l . ,
incompletely
type
Chinese
ovary
MM ( s e e
F i g . 2)
F i g . 2) acting
and the is
one
summarized
of
con-
acting
on
desig-
glycopeptides
w h i c h we d e s i g n a t e absence
It
cells
w h i c h we on
to
glyco-
glycosylated.
hamster
i n d i c a t i n g the presence
cells
the
1975;
resistant
lectin-binding
were
and the other
cells
to
c l a r i -
phytohemagglutinin-resistant
lectins
wild
MGn ( s e e
i n lectin-resistant et
f i r s t
structure
GlcNAc-transferase
I
structure.
GlcNAc,
two N - a c e t y l g l u c o s a m i n y l t r a n s f e r a s e s , with
the
Narasimhan
this
addition
will
The mutant
surface
that
nate
ferases
as
ovary
several
with
transferase
the
F i g . 2
hamster
shown
glycopeptides
catalyzing
a l . , 1977).
action
now b e e n
of
(MM, s e e
Chinese
Narasimhan
of
sugars:
section.
structure of
four
apparatus,
addition
core
mutant
elongation
of
and F u c ; t h e enzymes
located the
the
involves
of
GlcNAc-
both
trans-
GlcNAc-transferase
i n Table
I
(data
1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
from
30
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES The
were
derived
Fig.
2).
glycopeptides from
Glycopeptide
myeloma
structure
on
was
glycosidase by
A.
to
and
important
Strecker
MM.
The
et
was
of
of
We
ted
can
that
enzyme
into
thus
state
1977). than
controls
aid
31,2
data
linkage t o
enzyme
3-N-
t o
reaction
the
also
I t is,
add
Man-al,3absolute
shown
action
(Ito
ejt
adhered
than
into
Schachter, likely
in
Fig.
Both
depend
the
2),
sialic
residues. mutant and
however,
in
resistance
we
have
i t i s not
certain
the
Man-al,6-
terminus.
was
determined
on
by
subsequent
I i s MGn
GlcNAc-
above
leads
only had
yet
on any
linkage above The
for
addition
action
o fGal
and
surprising that
the
Chinese
hamster glycoprotein
lectins. MGn
(Narasimhan
GnM a v a i l a b l e
e t a l . ,
for
testing
I I i s specific
synthesized
i s 31/2;
GlcNAc-transferase
product
o f the
reaction
for this
I i s there-
2).
relationship
described
above
petition)
studies
between
i spresently have
the
under
indicated
two
study.
that
I
o f the
o fGlcNAc-
t o incomplete
i fGlcNAc-transferase
1977).
the
addition
not
t o several
I I acts
The
GlcNAc-transferase
lectin-resistant
not
asdescribed
e ta l . ,
(Fig. The
the
described
and
(Narasimhan
a s does
residue
I t i s therefore
GlcNAc-transferase
that
(Narasimhan
process.
cell
preparation).
a preferred
elongation
glycosylation
in
path.
o f a GlcNAc
enzyme
Man-al,6-
the
2 i s thus
entire
o fthis
action
incorpora-
available
the
acid
been
t oa Man-al,6-
residue i s not
product
1975); w e t o the
o f GlcNAc-transferase
addition
(Fig.
prepara-
o f C I . p e r f r i -
a l . ,
GlcNAc has
in
o f the
resistant
however,
a GlcNAc
and o f Fuc
GnGn
CI
and
terminus
pathway
Schachter,
t o the
rather
product
GnM.
and
susceptibility
the
Narasimhan
residue
fore
the
a l l
terminus
that
The
an
that
I
1977);
the
in
The GlcNAc
ovary
o f NMR
i s completely
transferase absence
with
o f the
I reaction
product
than
I can
i f the
a l . ,
next
residue
was
susceptibility
Wilson
studied
indicating
rather
terminus rather
the
Man-al,3-
the
transferase et
product
Narasimhan,
recently
o f MM ( W i l s o n ,
2)
(Fig.
Genetics, acid
t ob
1975;
have
found
terminus We
The
GlcNAc-transferase
this
sialic
and
carried
branch point
a GlcNAc by
endo-3-N-acetylglucosaminidase
ngens have
linkage
ajL. ,
ejt
the
spectra
t oConcanavali
GlcNAc-Man tion) .
I adds
determined
acetylglucosaminidase.
Ogata
220
and by
(1977a).
GlcNAc-transferase
strongly
c a r r i e d out
I t s
sequential
o fMedical
o f the
I
multiple
columns.
1977)
Department
Table (see
subsequent
resonance
Man-al,3-
a t the
and
analysis,
e t a l . ,
magnetic
in GS
a human
filtration
assignment
a n d was
a l .
3-linkage
from
digestion
gel
shown
structure
carbohydrate
Carver, The
N-acetyllactosamine
from
and
by
assays
the
obtained
proton
J . P .
the
with
(Narasimhan
resolution
o fToronto.
especially
for
pronase
exchange
established
Grey
University
ion
Gby
digestions
360 MHz h i g h out
GS w a s
immunoglobulin
purification
used
a glycopeptide
GlcNAc-transferases Mixed
substrate
MM a n d M G n c o m p e t e
for
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
(coma
2.
SCHACHTER ET AL.
common this
enzyme
finding
catalytic I
and
I
would
is
active does
have
(Narasimhan
disprove
a common t o have
i nthe
aJL.
biosynthesis
(1977)
in
wild
type
and
cells.
I nw i l d
type
cells,
virus
carries
(3 m a n n o s e
virus
i nthe
acetyllactosamine from
the
mannose
I
residues
per
processing
absence
one
possibility
As can
2).
(Fig. buted
The
usually enzyme
assayed was
using
into
the
rather
than
(Wilson
and
transferase (see
F i g .
Kessel with
adding
Fuc
because This
not
Thus,
i t cannot
scheme
saccharides
occur
t o the
acetyllactosamine-type others
donot
but
the
fucosyltransferase Perhaps ferase
i s the
to
terminal
the
related, lactose
this
i f not
More shown
can
act
on
Fuc
residue
t o the a l . ,
GnGn
near
the
process
I has
acted.
Asn-GlcNAc a Fuc
fucosyl-
MGn a n d
i s added
o f the
work
the
oligo
why
some N -
residue
while
appropriate
possibility.
thoroughly
et.
recent
know
with
cleavage
that
both
donot
the
residue
migrated
Golgi-localized
have
a^-acid that
product by
Fuc
has
o r absence
GlcNAc o f GnGn. (Brew
the
and
and i s
GlcNAc
endoglycosidase
we s t i l l
a likely
identical,
d i s t r i
Munro
1977)
internal
analyzing
oligosaccharides
most
1975;
showed
GlcNAc-transferase
galactosyltransferase
synthetase
most
Fuc
residue
i s widely
(1976)
radioactive
after
presence
seems
the
and CI;
Fuc;
2),
(Fig.
Chou e t a l . ,
oligomannoside-type
contain
subunit o f
Asn
e t a l . ,
e t a l .
i s a late
until
known;
β-galactosidase-treated
o n MM b u t
why
the
oligosaccharide.
addition
explains never
5
between
i s not
t o MM
reaction
Munro
i np r e p a r a t i o n )
although
connection
regulatory
nearest
1977;
acceptors
act
The
added
this
Wilson
uncharged
chain,
virus
(about
processing.
been
sialidase-,
Schachter, w i l l
expected
i n removing
1971;
Asn-GlcNAc moiety with
absence o f mature
during oligosaccharide
e t a l . ,
as acceptor.
2).
polypeptide
cells.
i s somehow
catalyzing
glycopeptide
charged
the the
I and p r o c e s s i n g
GlcNAc
Schachter,
i nfact
than
However,
s i a l y l - N -
involved
endo-3-N-acetylglucosaminidase the
mannose
postulated
above)
residues
enzyme and
from
Surprisingly,
more
a s a GlcNAc has
1973;
glycoprotein
the
(see
mannose
(Jabbal
Schachter,
by
I
be incorporated
oligo
lacks
bepredicted
I-deficient
i s that
soon
line
ovary
o f the
oligosaccharide).
2).
F i g .
o f GlcNAc-transferase
two
hamster
glycoprotein
c e l l
studied glyco
(se
the
last
have
envelope
Chinese
which
oligosaccharide)
GlcNAc-transferase
the
per
as would
carries
the
GlcNAc-transferase
virus
N-acetyllactosamine-type
(see
cells
subunit above.
(1978)
e ta l .
envelope
residues
arms,
mutant
saccharide
the
GlcNAc-transferases GlcNAc-transferase
described
stomatitis
lectin-resistant
GlcNAc-transferase
that
different
regulatory
mutant
Although
o f two
subunit;
lectin-resistant
only
saccharides grown
suggests
and Tabas
o f vesicular
1977) .
e t a l . ,
catalytic
31
and Catabolism
existence
an additional
protein mature
the
lectin-resistant
Hunt e t the
Biosynthesis
i t nevertheless
share
then
absent
site
not
sites,
I I may
Glycoprotein
characterized which
This
adds
enzyme
A protein 1968) .
The
Gal
glycosyltransi n 31/4
i s believed component A protein
linkage
t o be
o f milk has
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
a
very
32
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
low
affinity
for glucose;
lactalbumin),
affinity
lactose
is
mammary
gland,
A
synthesized.
protein
can
lactose
(or a very
incorporate
terminal
i n the presence
for glucose Since
Gal into
glycoproteins glycoprotein
absence
fetuin,
linkage
of
Asn-GlcNAc oligosaccharides.
α-lactalbumin and r a t serum
homogeneity
by c l a s s i c a l
by
affinity
chromatography
or
UDP-hexanolamine-Sepharose
Andrews, and
1970;
Brew,
active and
1974;
Fraser
bovine
forms
with
that
smaller
due t o
1976). enzyme
of
molecular
be
41,000 b y been
to
from
milk
enzyme
is
the
the
enzyme
elongation
milk,
et
a l . , 1970)
et
and H i l l ,
to and
1971;
a l . , 1976;
51,000
potent
Powell
larger
that
a
be
protease molecule
is
form
milk
single
by
form
lactating
X-100
of
of
enzyme
weight has
sheep
colostrum
also mammary
fat
gland
enzyme
and, on p u r i f i c a t i o n ,
showed
molecular
molecular
t h e mammary It
the
bovine
this
t h e mammary
e_t a l . ,
of
molecular
and from bovine
proteo
from
inhibitors;
of
inactibelieved
(Magee
isolated
with
t h e two
heat
Galactosyltransferase
enzyme.
produced
the
could
component of
55,000-59,000
and i t
i n bovine
et_ a l . , 1 9 7 7 ) ;
component
be
substrate,
reagents
membranes
1977)
to
et. a l . , 1 9 7 4 ) ;
for
found
1% T r i t o n
the milk
M
from
trypsin.
properties of
of
purified
estimated
protease
smaller
a major
and a minor
K
derived
(Powell
components,
properties
a^-acid
a n d human
(Trayer
(Magee
to
(1974)
and Brew,
two
enzymic
a of
solubilized with
69,000
weights
the Golgi
was
The
is
contains
membranes
GlcNAc-
a-lactalbumin-Sepharose
Geren
sulfhydryl
weight
the action
(Smith
globule
columns
regard
and Brew
which
isolated
glands
form
degraded
GlcNAc,
been
(Fitzgerald either
The
and GlcNAc-terminal
certainly
have
i n
d i s t r i b u t e d and
The f u n c t i o n
The bovine
respectively
molecular
colostrum could
free
enzymes
using
a plasmin-like
Powell
to
almost
a l . , 1974;
and i n h i b i t i o n by
the
lysis
widely
only
organ.
an
are similar with
vation
present
i n this
(a-
and
milk
42,000-44,000,
forms
is
methods
Khatra et
The
is
only
3-galactosidase-treated
the
colostrum
increased
is
for example).
in
bovine
of
the Β protein
and glycopeptides,
(sialidase-, or
occurs
protein)
81/4
oligosaccharides
of
greatly
α-lactalbumin
synthesis
similar
is
was
by p r o t e o l y s i s
weight
weight
gland of
53,000-55,000.
enzyme
suggested the
65,000-
resembled
that
the
the
soluble
Golgi-bound
transferase. The
various
glycoproteins 1977;
Lehman e t of
this
addition
of
Mn2+,
et
studied et
enzyme
the galactosyltransferase
about
have
Bell
a l . , 1974;
1973; et
10-15%
Trayer been
UDP-galactose,
(Ebner,
a l . , 1975a,b;
Khatra
of
a l . , 1975;
kinetics been
forms
and contain
carbohydrate
are
(Geren
and H i l l ,
1971).
published
and the order
α-lactalbumin,
Tsopanakis
a l . , 1976;
Powell
of
and acceptor
and Herries,
K i t c h e n and Andrews,
et a l . ,
Detailed
1976;
and Brew,
have
Geren
1975;
1974a,b).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL. The
a-1,6There
scheme
terminus is
no
in
F i g .
prior
direct
to
evidence
preferntial
both
bovine
immunoglobulin
myeloma
immunoglobulin
communication) GnG.
Also,
transferase
that
specificity.
contain
Rao
depicts
addition
such ple
2
Biosynthesis
e_t
the
a l .
purified
ferred
Gal
more
rapidly
G than
to
Elongation residue
to
various
Asn-GlcNAc
primarily reports have
GG a2,3
of
and
other
reported
glycoprotein
is
(Fig.
a
tissues acid
into
Roden,
interesting,
G
(Tai
et
al_.,
G
(Baenziger have
swine
(Montreuil,
Isemura shown of
assumed
that
measuring
(Bartholomew
further, were the
et
of
rat
1977b)showed
which
makes
sialic is
acid
evidence
may
be
incorporation (see
section i n
s i a l i c
acid
into
to
transferase two
forms
by
and of
a l . ,
to
be
occasional
a2,3 into
of
acid
et_ a l _ . from
1972). et
preparations,
and
1972)
Support
rat,
pork,
catalyze
two
the
However,
probably
1977;
not
of
sialidase-
Van
den
the
Eijnden
was
a2,6;
transferase
involved
in
oligosaccharides.
this
i n
analysis
on
synthesized Thus,
There
3 -sialyltransferase 1
incorporation transferases
activities
sialyltransferases
action
a l . ,
Asn-GlcNAc that
and
recently
into
which i n
Ser(Thr)incorporate
linkages
other
discovered. (1977a) of
have
recently
CMP-sialic acid:
bovine
affinity
equal
until
colostrum
these
colostrum.
chromatography
on
above,
specific
this
reported enzyme
with
p u r i f i -
was
a2-6
purified
CDP-hexanolamineLike
the
sialyltransferase
activity,
the
3-D-galactoside The
CDP-ethanolamine-Sepharose.
discussed
CMP-sialic
(Schachter
separate
linkage.
V below)
s i a l y l -
sialyl-a2,6-lactose;
linkage
is
a
Various
from
1977b)
and
two
only
The
be
homogeneity
sialyltransferase Sepharose
crude
Asn-GlcNAc oligosaccharides
remain
440,000-fold
reported
membrane
Schachter,
(Stoffyn
the
had
crude
et
and
that
sialyl-a2,3-lactose
Paulson
acid
i n
suggested
acid
It
that
Schachter,
indication of
involved
to
been
have
sialyltransferase
that
sialic
cation
immuno-
sialyltransferase.
development
glycoprotein
oligosaccharides.
a2,6
p-
trans-
s i a l i c
protein.
s i a l i c
one
Paulson
and
GalNAc than
a
Golgi-localized
using
(Hudgin
liver
oxacid no
of
glycoprotein
finding
indicated
treated
was
a
sialyl-a2,3-lactose
et_ a l . , there
by
linkages
however,
same
1978).
than
the
1973;
(Hudgin
of
than
galactosyl-
porcine
have
(1971)
the
a^-acid
assays,
liver
liver
involved product
for
contain
more
differential
rat
Schmid
1974a,b,
from
a l . ,
both
the
embryonic
to
these
came
a n d human
synthesis
a
glycopeptides.
1975);
incorporating
Schachter,
i n
bovine
and
linkage
sialidase-treated
idea
personal
nodes
addition
oligosaccharides
been
this
from
that multi-
rather
that
acid-galactose
type
were for
2)
lymph
shows
human
chromatography
GnGn p r e p a r e d the
and
(Fig.
reported
by
Man-
however,
and K o r n f e l d ,
mesentary
Sialic
the
linkage
been
fact
1975)
GGn
affinity
completed
to
terminus.
galactosyltransferase
a2,6
capable
1973;
Gal
sialyl-a2,4-galactos
and
have
of
Man-a-1,3-
GlcNAc-terminal
2).
sialyl-a2,2-galactose transferase
the
structure
to
other
the
is
aminophenyl-8-GlcNAc-Sepharose globulin
addition
to
33
Catabolism
It
(1976)
from
and
molecular
galactosylwas
found
weights
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
in of
34
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
56,000 that
and 43,000
the
larger
smaller
enzyme.
might
as
observed
crude
enzyme
enzymes
were
with
1973).
Both
ot^-acid
glycoprotein,
sialic
acid-free
termini
whereas
purified as
the latter
above,
t h e a2,6
both We
have
crude
shown
described
above
and pork
liver
of
tissues
Although
organelles the
that
appears i n which
into
the
fuses
a
with
applied
on the site
cells
of
membrane
is
with
within
towards
F i g . 3). membrane by
evidence
comes
radioactivity o r by
auto
and Leblond,
the Golgi
1977).
apparatus,
the plasma
membrane
The v e s i c l e
membrane
glycoproteins
glycoproteins
a
,
Golgi
radioactive
techniques
Bennett
cells)
the
incorporation of
1978;
membrane
1974a,b,
subcellular
certain
Supportive
and secretory
whereas
lateral
are
probably
diffusion
process.
(threonine)-N-acetyl-D-galactosamine
similar
to
on the synthesis
studies
into
to
those
Almost
glycoproteins
two o v e r - l a p p i n g of
mucins of
with
blood
discuss
glycoprotein
the highly
active
specialized
(Watkins,
1978).
A brief
maxillary
mucin biosynthesis involved
1974;
discussion
i n mucin
the
mucins,
will
of
serve
has
since ovine to
synthesis glands tracts,
of
the
and
i n particular
various
will
be group
detailed
and Roden,
and porcine
illustrate
(Fig.
dealt
human b l o o d
Schachter of
synthesis
been
classified
No a t t e m p t
field
and g l y c o l i p i d biosynthesis
are available
(i)
a n d mucous
antigenicity.
Schachter principles
cells
work
and g e n i t o - u r i n a r y
group
human A B O - L e w i s
have
and can be
categories:
(ii)
above
Ser(Thr)-GalNAc-linkage
"mucins"
by goblet
respiratory
the synthesis
described of
a l l the reported
or
gastro-intestinal,
reviews
of
Approaches
secretion
to
of
elongation
are pulsed
migrates
serine
secretory
made
surfaces of
completed (see
Schachter
oligosaccharides.
oligosaccharides.
mucins
liver
sialyltransferase
i n other
by biochemical
is
the plasma
type
and
and
a l . , 1974; may o c c u r
1974a,b,
the c e l l
with
broadly
et
vesicle
the plasma of
and
sialyl-lactose.
N-acetylglucosaminyltransferases,
a r e made
either
glycoprotein
Biosynthesis type
the
of
conclusive.
intact
elongation
from
become p a r t V.
highly
transport
extruded
The
colostrum
enzymes
(Schachter
finished
within
termini.
from
isomers
(Letts
and measurements
Once
Gal-$1,3-GalNAc
(Munr
glycoproteins
radiography
various
Gal-βΐ,4-GlcNAc
preparations
the major
from
sugars
acid-free
sialyl-a2,6-lactose;
that
these
apparatus work
been
and Roden,
only
(in particular,
evidence
$-
had previously sialic
contain
for
with G a l -
are highl
and
1978).
former
galactosyltransferase
rat
other
with
shown, the
synthesizes
a n d a2,3
fucosyltransferase,
and other
(Schachter
active
of
specific
IgG and IgM but not w i t h
have
sialyltransferase
mentioned
make
highly
fetuin, the
highly
specificities
preparations
but not
product
low a c t i v i t y
Gal-31,4-Glc
substrate
mucins;
were
and showed
Gal-$l,6-GlcNAc, similar
a degradation
enzymes
an acceptor
galactosides;
i t was p o s t u l a t e d ,
be
Both p u r i f i e d
Gal-$l,4-GlcNAc 31,3-GlcNAc,
respectively; enzyme
the
4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1973; sub general
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
35
and Catabolism
TABLE I N-acetylglucosaminyltransferase l e v e l s of w i l d type and l e c t i n r e s i s t a n t Chinese hamster ovary c e l l s using as acceptors glycopeptides prepared from human IgG; see F i g u r e 2 for glycopeptide nomenclature. IgG glycopeptide acceptor
N-acetylglucosaminyltransferase activity (nmol/mg/hour) Wild type cells
MM
Lectin-resistant cells < 0.2
3.7
MGn GnGn
R
-0H
<0.2
<0.2
U D p
-
G o l N A c
, R-0-GalNAc
CMP-SIOUC
Acid
(POLYPEPTIDE CORE)
,
R
.
Q
.
G
a
L
N
A
c
.
S
A
(OSM) UOP-Gal
R - Ο GalNAc I Gal
CMP-SA (?)
-R-O-GalNAc-SA Gal ι
ODP-Fuc I
CMP-SA (?)
R-0-GalNAc I Gal-Fuc
•R-O-GalNAc-SA Gal—Fuc (A")-PSM ι
UDP-GalNAc
R-0-GalNAc
I
I (?)
CMP-SA (?)
Gal— Fuc I GalNAc
i -R-O-GalNAc-SA Gal—Fuc I GalNAc (A*)-PSM
Figure 4.
Assembly of ovine (OSM) and porcine (PSM) submaxilhry mucins
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
36
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
The f i r s t important p o i n t i s t h a t i n i t i a t i o n does not i n volve pre-assembly o f o l i g o s a c c h a r i d e as a l i p i d intermediate. I t has been reasonably e s t a b l i s h e d i n s e v e r a l systems t h a t the Ser(Thr)-GalNAc l i n k a g e i s synthesized by the i n c o r p o r a t i o n o f a GalNAc from UDP-GalNAc i n t o a high molecular weight acceptor. The t r a n s f e r a s e has a high s p e c i f i c i t y f o r acceptor; f o r example, the enzyme from s a l i v a r y gland w i l l donate GalNAc t o mucin t r e a t e d with g l y c o s i d a s e s t o remove a l l carbohydrate but not t o a l a r g e number o f other high and low molecular weight acceptors. An i n t e r e s t i n g exception i s the t r a n s f e r o f GalNAc t o a b a s i c p r o t e i n i s o l a t e d from bovine myelin (Hagopian e t al_., 1971) ; t h i s p r o t e i n i s not normally a g l y c o p r o t e i n and the s i g n i f i c a n c e o f i t s acceptor a c t i v i t y i s not known. Although pronase treatment of carbohydrate-free mucin acceptor destroys acceptor a c t i v i t y , a recent r e p o r t has shown t h a t the enzyme w i l l t r a n s f e r GalNAc to t r y p t i c peptides o f carbohydrate-free ovine submaxillary mucin ( H i l l e t a l . , 1977) s e r i n e and threonine residue saccharides have i n d i c a t e d no homologies t o d e f i n e the substrate s p e c i f i c i t y o f the t r a n s f e r a s e (Marshall, 1974; H i l l e t a l . , 1977). The enzyme r e q u i r e s high molecular weight acceptors but i t i s not c l e a r why some hydroxyamino a c i d s are g l y c o s y l a t e d but others are not. I t i s i n t e r e s t i n g t h a t the porcine submaxillary gland UDP-GalNAc:polypeptide N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e does not adhere t o UDP-hexanolamine-Sepharose and has thus f a r been p u r i f i e d only 3 0 - f o l d ; however, t h i s p r e p a r a t i o n was r e l a t i v e l y f r e e o f other mucin g l y c o s y l t r a n s f e r a s e s ( H i l l e t aJL., 1977) . There i s an important branch p o i n t immediately a f t e r GalNAc i n c o r p o r a t i o n . I f s i a l i c a c i d i n c o r p o r a t i o n occurs t o form the sialyl-a2,6-GalNAc d i s a c c h a r i d e , f u r t h e r carbohydrate i n c o r p o r a t i o n ceases and the predominant form i n ovine sub m a x i l l a r y mucin r e s u l t s ( F i g . 4 ) . I f , however, Gal i s i n c o r p o r a ted to form the Gal-βΐ,3-GalNAc d i s a c c h a r i d e , the various o l i g o saccharides present i n p o r c i n e submaxillary mucin r e s u l t (Fig. 4 ) . The r e l a t i v e p r o p o r t i o n s o f the s i a l y l - and g a l a c t o s y l t r a n s f e r a s e s c o n t r o l t h i s branch p o i n t (Schachter et_ al^. , 1971; McGuire, 1970). The C M P - s i a l i c acid:GalNAc-mucin a2-6 s i a l y l t r a n s f e r a s e has been p a r t i a l l y p u r i f i e d from sheep submaxillary gland (Carlson e t a l . , 1973 ) and porcine submaxillary gland (Sadler e t a l . , 1977). The b e s t substrate f o r t h i s enzyme i s s i a l i d a s e - t r e a t e d ovine submaxillary mucin (GalNAc-mucin) and other s i a l i c a c i d - f r e e mucins. The enzyme does not a c t on s i a l i d a s e - t r e a t e d αι-acid g l y c o p r o t e i n nor on l a c t o s e and i s t h e r e f o r e d i f f e r e n t from the s i a l y l t r a n s f e r a s e s d i s c u s s e d i n the previous s e c t i o n ; a v a r i e t y o f low molecular weight compounds both with and without t e r m i n a l GalNAc were i n e f f e c t i v e as acceptors although Gal-81/3-GalNAc had low acceptor a c t i v i t y . A n t i f r e e z e g l y c o p r o t e i n , which contains many Gal-81r3-GalNAc u n i t s , i s an e x c e l l e n t acceptor (Sadler e t a l . , 1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and
Catabolism
37
The key enzyme i n c o n t r o l l i n g s y n t h e s i s appears t o be the UDP-Gal:GalNAc-mucin 81-3 g a l a c t o s y l t r a n s f e r a s e (Schachter et a l . , 1971). This enzyme i s t i g h t l y bound t o membrane and a l l attempts a t p u r i f i c a t i o n have been u n s u c c e s s f u l . The enzyme w i l l not a c t i f GalNAc i s s u b s t i t u t e d with a s i a l i c a c i d residue (Fig. 4 ) . However, i f Gal i s i n c o r p o r a t e d , other sugar residues can be added. Thus, s i a l i c a c i d can then be added t o the i n t e r n a l GalNAc. F u r t h e r , Fuc can be i n c o r p o r a t e d i n a l , 2 linkage t o the Gal r e s i d u e ; t h i s enzyme i s s i m i l a r t o the human blood group H f u c o s y l t r a n s f e r a s e and can t r a n s f e r Fuc t o a v a r i e t y o f high and low molecular weight compounds with a t e r m i n a l 8-galactoside residue (McGuire, 1970) . The f u c o s y l t r a n s f e r a s e has r e c e n t l y been p u r i f i e d to near homogeneity from porcine submaxillary glands using GDP-hexanolamine-Sepharose (Beyer e t aJL. , 1977) . I t i s i n t e r e s t i n g t h a t although the f u c o s y l t r a n s f e r a s e w i l l a c t on a v a r i e t y o f 8 - g a l a c t o s i d e s i t p r e f e r s Gal-81,3-GalNAc Beyer e t a l . , 1977). The f i n a l enzyme i n the porcine submaxillary mucin syn t h e s i s scheme i s UDP-GalNAc:Gal-mUcin N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e . This enzyme i s analogous t o the human blood group Α-dependent N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e and occurs only i n those p i g s g e n e t i c a l l y disposed towards making A - p o s i t i v e mucin. The enzyme i n c o r p o r a t e s GalNAc i n a l , 3 linkage t o the Gal residue o f F u c - a l , 2 - G a l - t e r m i n a l acceptors. The p o r c i n e submaxillary gland enzyme was p u r i f i e d 38,000-fold by a f f i n i t y chromatography on UDP-hexanolamine-Sepharose (Schwyzer and H i l l , 1977a) and d e t a i l e d k i n e t i c s t u d i e s have been c a r r i e d out (Schwyzer and H i l l , 1977b). The pure enzyme has a molecular weight o f about 100,000 and may c o n t a i n 2 subunits. I t i s c l e a r from the above t h a t mucins are assembled by step-by-step a d d i t i o n o f monosaccharides; no pre-assembly seems to occur. The s u b c e l l u l a r path o f b i o s y n t h e s i s has not been worked out i n as great d e t a i l as f o r Asn-GlcNAc o l i g o s a c c h a r i d e s but passage through the G o l g i apparatus probably occurs (Bennett and Leblond, 1977; Schauer e t a l . , 1974). Glycophorin and other g l y c o p r o t e i n s have been reported to c a r r y a sialyl-a2,3-Gal-81,3-(sialyl-a2,6)-GalNAc t e t r a s a c c h a r i d e s t r u c t u r e (Marchesi e t a l . , 1976). The s i a l y l t r a n s f e r a s e c a t a l y z i n g t h i s a2,3 linkage t o galactose i s probably the same enzyme t h a t c a t a l y z e s s y n t h e s i s o f s i a l y l 0.2,3-lactose ; the s y n t h e s i s o f the a2,3 and a2,6 isomers o f s i a l y l - l a c t o s e by colostrum and l i v e r were d i s c u s s e d i n the p r e vious s e c t i o n . Sadler e t a l . (1977) p u r i f i e d two d i s t i n c t s i a l y l t r a n s f e r a s e s from porcine submaxillary gland by the use o f CDP-hexanolamine-Sepharose a f f i n i t y chromatography; one enzyme was the C M P - s i a l i c acid:GalNAc-mucin a2-6 s i a l y l t r a n s f e r a s e d i s cussed above, the other was an enzyme a c t i n g on a v a r i e t y o f acceptors to make a s i a l y l - a 2 , 3 - G a l l i n k a g e . Among the acceptors found a c t i v e with the l a t t e r enzyme were l a c t o s e , G a l - 8 1 , 3 -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
38
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
9?
S-a2,6-G-Bl,4-Gn-Bl , 4 . . * S-a2,6-G-Bl,4-Gn-Bl,2-M-o
^y-el,4-Gn-el> S-a2,6-G-Bl,4-Gn-Bl,2-M-al,6 Gn
|n-Asn
61,4> S-a2,6-G-Bl ,4-Gn-Bl ,4*'
<sf 0 Θ DEFICIENT ENZYME
IN-BORN ERROR
4-L-aspartylglycosylamlne amldohydrolase (enzyme 1)
Aspartylglycosylamlnurla (AGU)
a-mannosldase (enzyme 3) 8-N-acetylhexosamlnldases A and Β (enzyme 4)
STRUCTURES ACCUMULATING
Gn-Asn M-cl,6-M-el,4-Gn-el,4-Gn-Asn
M-ol,3-M-el,4-Gn
GL,,-gangl1os1dos1s variant 0 (SandhoffJatzkewltz disease)
Gn-Bl,2-M-al,3-Μ-βΙ,4-Gn
n c
Gn-6l,4-M-al,3-M-el ,4-Gn
Pollltt and Pretty (1974) Pollltt and Jenner (1969) Lundblad et a l . (1976) AkasaM et aTT (1976) Norden et al.. (1974)
Strecker et a l . (1977b) Ng Ylng KTn and Wolfe (1974)
Gn-Bl,2-M-al,3 v Gn-βΐ,4-Μ-βΙ,4-Gn Gn-Bl ,2-M-al , β ' Gn-Bl ,4 y Gn-βΐ,2-M-al,3 or 6 Gn-Bl,2-M-ol ,6 or 3
B-galactos1dase (enzyme 5)
(L,,-gangliosidosis ™
G-Bl,4-Gn-el,4^
Ng Y1ng K1n and Wolfe (1975)
G-Bl,4-Gn-Bl,2-M-ol,3 or 6
Wolfe et ai. (1974)
* M-βΙ,4-Gn G-Bl,4-Gn-el,2-M-al ,6 or 3 S1al1dase
Mucolipidosis I
(enzyme 6)
Mucolipidosis II (I Cell Disease)
S-a2,6-G-Bl,4-Gn-Bl,2-M-al,3
Michel ski et a l . (1977) Strecker et al_. (1976)
S-a2,6-G-Bl,4-Gn-Bl,2-M-al,6
Figure 5. Scheme depicting accumulation of glycopeptides and oligosaccharides derived from Asn-GlcNAc oligosaccharides in the urine and organs of patients with in-born errors of metabolism involving the absence of glycosidases. A typical Asn-GlcNAc oligosac charide of the N-acetyuactosamine type is shown at the top of the figure. Abbreviations: S, sialic acid; G, Ό-galactose; Gn, N-acetyl-O-glucosamine; M, Ό-mannose, F, \,-fucose; Asn, asparagine.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
GalNAc,
antifreeze
submaxillary an
enzyme
glycoprotein
mucin.
capable
Pork
to
a sialyltransferase
GalNAc-Ser(Thr) least
saccharides. as
these
These
Many
organisms, variety
It
i s
will
porcine contain
(Hudgin and
has also
been
shown
sialyl-a2,3-Gal-81,3-
making
Therefore,
a c t on Ser(Thr)-GalNAc type act not only
on secretory
o n membrane-bound
glyco-
glycoproteins
therefore
reasonable
be widely
distributed.
at
oligo-
to predict
such
that
tides
limitations
plants
have
and both
which
t o a group
storage
of
appear
to be derived 5
Fig.
summarizes
the
exoglycosidases
end
one a t a time.
prevents
the action
apparent
that
known.
of
this
i n micro-
and vertebrate
animals
i n organs
and urine
from Asn-GlcNAc
normally
remove
The absence
briefly
of
sugars
I t
from
next
of action
process
o f these i n the
It
i s
(enzyme
also No.2,
are required
backbone.
diseases
that
exoglycosidase
N o . l , F i g . 5)
from polypeptide
located
i s evident
i n line.
o f the exo- and endo-
studies i s
(enzyme
d i s -
structures
the non-reducing
of a particular
o f t h e enzyme
of
glycopepsome
oligosaccharides.
data.
endo-8-N-acetylglucosaminidases
Pathological
degradation
consider
some o f t h i s
oligosaccharide
sequence
mucopolysaccharides,
We w i l l
and an asparaginase
separate
exact
discussion
the presence
o
glycolipids,
involving accumulation
5)
extensive
shown
invertebrate
andoligosaccharides.
orders
Fig.
preclude
laboratories
o f e x o - and endo
relevant
involving
to
I V above)
to
Glycoprotein Catabolism. Space
ly
enzymes
but also
two enzymes
topic. a
shown
( V a n d e n E i j n d e n e t a l . , 1977a)..
(mucins)
glycophorin.
VI.
section
two s i a l y l t r a n s f e r a s e s
proteins
previously
39
Catabolism
sialyl-a2,3-lactose
o f making
1972;
see also
and
and s i a l i d a s e - t r e a t e d
liver,
Schachter, contain
Biosynthesis
enzymes indicate
The i s not that
lysosomes.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
the
40
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
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44
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Powell, J.T. and Brew, K. (1975). J. Biol. Chem. 250, 6337-6343. Powell, J.T., Jarlfors, U. and Brew, K. (1977). J. Cell Biol. 72, 617-627. Rao, A.K., Garver, F., and Mendicino, J. (1976). Biochemistry 15, 5001-5009. Robbins, P.W. (1977). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Rothman, J.E. and Lodish, H.F. (1977). Nature 269, 775-780. Sadler, J.E., Rearick, J.I., Paulson, J.C. and Hill, R.L. (1977). Proc. International Symp Glycoconjugates Wood Hole Mass., in press Sato, T., Yosizawa, Z., Masubuchi, M. and Yamauchi, F. (1967). Carbohyd. Res.5,387-398. Schachter, H. (1974a). Adv. Cytopharmacol. 2, 207-218. Schachter, H. (1974b). Biochem. Soc. Symp. 40, 57-71. Schachter, H. (1977). In "Mucus in Health and Disease" (M. Elstein and D.V. Parke, editors), pp. 103-129, Plenum Publish. Co. Schachter, H. (1978). In "The Glycoconjugates" (M. Horowitz and W. Pigman, editors), Vol. 2, Academic Press, New York, in press. Schachter, H. and Roden, L. (1973). In "Metabolic Conjugation and Metablic Hydrolysis" (W.H. Fishman, editor), Vol. 3, pp. 1-149, Academic Press, New York. Schachter, H., Jabbal, I., Hudgin, R.L., Pinteric, L., McGuire, E.J., and Roseman, S. (1970). J. Biol. Chem. 245, 1090-1100. Schachter, H., McGuire, E.J. and Roseman, S. (1971). J. Biol. Chem. 246, 5321-5328. Schauer, R., Buscher, H.-P., and Casals-Stenzel, J. (1974). Biochem. Soc. Symp. 40, 87-116. Schwyzer, M. and Hill, R.L. (1977a). J. Biol. Chem. 252, 2338-2345.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
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Glycoprotein
Biosynthesis
and Cataholism
45
Schwyzer, M. and Hill, R.L. (1977b). J. Biol. Chem. 252, 2346-2355. Sherr, C.J. and Uhr, J.W. (1969). Proc. Nat. Acad. Sci. USA 64, 381-387. Smith, C.A. and Brew, K. (1977). J. Biol. Chem. 252, 7294-7299. Spiro, M.J., Spiro, R.G. and Bhoyroo, V.D. (1976a). J. Biol. Chem. 251, 6400-6408. Spiro, M.J., Spiro, R.G. and Bhoyroo, V.D. (1976b). J. Biol. Chem. 251, 6420-6425. Spiro, R.G., Spiro, M.J d Bhoyroo V.D (1976) J Biol Chem. 251, 6409-6419 Stanley, P., Narasimhan, S., Siminovitch, L., and Schachter, H. (1975). Proc. Nat. Acad. Sci. USA72,3323-3327. Stoffyn, P., Van den Eijnden, D. and Stoffyn, A. (1977). Fed. Proc.36,744. Strecker, G., Hondi-Assah, T., Fournet, B., Spik, G., Montreuil, J., MarOteaux, P., Durand, P., and Farriaux, J-P. (1976). Biochim. Biophys. Acta 444, 349-358. Strecker, G., Fournet, Β., Spik, G., Montreuil, J., Dorland, L., Haverkamp, J., Schut, B.L. and Vliegenthart, J.F.G. (1977a). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Strecker,G.,Herlant-Peers, M-C, Fournet, Β., Montreuil, J., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., and Farriaux, J-P. (1977b). Eur. J. Biochem. 81, 165-171. Struck, D.K. and Lennarz, W.J. (1977). J. Biol. Chem. 252, 1007-1013. Tabas, I., Schlesinger, S. and Kornfeld, S. (1978). J. Biol. Chem 253, 716-722. Tai, T., Ito, S., Yamashita, K., Muramatsu, T. and Kobata, A. (1975). Biochem. Biophys. Res. Communs.65,968-974. Tonneguzzo, F. and Ghosh, H.P. (1975). FEBS Lett.50,369-373. Trayer, I.P. and Hill, R.L. (1971). J. Biol. Chem. 246, 6666-6675.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
46
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Tsopanakis, A.D. and Herries, D.G. (1976). Biochem. Biophys. Res. Communs.68,1102. Turco, S.J., Stetson, B. and Robbiris, P.W. (1977). Proc. Nat. Acad. Sci. USA 74, 4411-4414. Van den Eijnden, D.H., Dieleman, B. and Schiphorst, W.C.M. (1977a). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Van den Eijnden, D.H., Stoffyn, P., Stoffyn, A. and Schiphorst, W.E.C.M. (1977b). Eur. J. Biochem. 81, 1-7. Vargas, V.I. and Carminatti, H. (1977). Mol. Cell. Biochem. 16, 171-176. Waechter, C.J. and Lennarz, 45, 95-112.
(1976).
Watkins, W.M. (1974). In "The Red Cell" (D.M. Surgenor, editor), 2nd edition, Vol. 1, pp. 293-360, Academic Press, New York; Biochem. Soc. Symp.40,125. Wilson, J.R., Williams, D. and Schachter, H. (1976). Biochem. Biophys. Res. Communs.72,909-916. Wirth, D.F., Katz, F., Small, B. and Lodish, H.F. (1977). Cell 10, 253-263. Wolfe, L.S., Senior, R.G. and Ng Ying Kin, N.M.K. (1974). J. Biol. Chem. 249, 1828-1838. RECEIVED April 7, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3 Structure and Metabolism of Glycolipids C. C. SWEELEY, Y.-K. FUNG, B. A. MACHER, J. R. MOSKAL, and H. A. NUNEZ Department of Biochemistry, Michigan State University, East Lansing, MI 48824
Glycolipids have bee some important biologica as contact inhibition, intercellular adhesion, immunochemical tissue specificity, hormone receptor function, and internalization of various external macromolecular and supramolecular materials. Close to a hundred different glycolipids have been isolated and characterized from various terrestrial and marine organisms to date, providing sufficient structural diversity for these proposed functional roles. Interest in the chemistry of these substances was stimulated by the discovery of a family of inherited abnormalities in the metabolism of some of the glycolipids (13). Subsequently they became a subject of intense study by cell biologists and immunologists when it was generally recognized that they are membrane constituents of most tissues; current areas of research interest are the immunochemical specificity of glycolipids, their localization and mobility in bilayer membranes, lectin-binding properties, and viral transformation-specific changes in their composition. The aim of this review is to provide an introduction into the chemistry, methods of analysis and metabolism of the major classes of glycolipids that occur in eukaryotic organisms. Structure Unlike the complex lipopolysaccharides of bacterial membranes or the glycosides of hydroxylated fatty acids in yeasts, the glycolipids of eukaryotic organisms are of two major types, called glycosphingolipids and glycoglycerolipids. Glycosphingolipids are composed of a family of longchain aliphatic bases (sphingosines), fatty acids and carbohydrates, whereas the glycoglycerolipids consist of glycerol, fatty acids (or fatty ethers) and carbohydrates. The glycosphingolipids can be subdivided into several classes on the basis of gross molecular features. Thus, there are neutral glycosphingolipids, gangliosides (glycosphingolipids containing sialic acid), sulfoglycosphingolipids (containing sulfate ester groups on the carbohydrate moiety) and phosphoglycosphingolipids. There are also neutral and sulfoglycoglycerolipids. Complete review of the structures and occurrence of the glycosphingolipids and glycoglycerolipids is not possible in this brief presentation. Recent reviews with more extensive 0-8412-0452-7/78/47-080-047$09.75/0 © 1978 American Chemical Society American Chemical Society Library In Glycoproteins and Glycolipids in Disease 1 1 5 5 1 6 t h St. N.Processes; W. Walborg,DC,E.;1978. ACS Symposium Series;W American ashingtChemical on, D. Society: C. 2CWashington, 036
48
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
information about specific topics are cited whenever possible to assist the reader with a need for more than a casual introduction to the field. An excellent text (4) on the isolation, structure analysis and metabolism of glycolipids, with an emphasis on details of methods and procedures, should also be consulted. Ceramide is the hydrophobic portion of a glycosphingolipid that is anchored in a bilayer membrane. It consists of a mixture of molecular species in which the long-chain bases such as sphing-4-enine are attached through amide linkages to several fatty acids. The most common longchain bases are shown below, but a variety of other chain-length homologs, branched sphingosines and multiply unsaturated types are known to exist (5). They are all derived from sphinganine, previously known as dihydrosphingosine, which is D-erythro-2-aminooctadecane-1,3-diol or 2S,3R-2-aminooctadecane-l,3-diol (6). Methods have been described for their analysis as aldehydes after periodate oxidation (7), N-acetyltrimethylsilyl derivatives (8), dinitrophenyl derivatives (9), and as the alcohols obtained from intac ozonolysis and reduction (10>) CHUOH
CH9OH
I
HC-NH
I
HC - OH
I
(CH9)..
I
2
CH-
1
*
3
I
CH9OH
I
HC-NH
9
I
I
9
HC-NH
I
I
9
1
H C - OH
H C - OH
HC
HC
II
II
I
I
CH
(ÇH2)12 CH^ Sphinganine
CH9OH
I
4-Sphingenine
CH
(ÇH2)W
HC— N H 9
I
HC -
I
OH
HC-OH
I
(CH9)n
I CH
3
CH^ 4-Eicosasphingenine
4-D-Hydroxysphinganine The fatty acids occurring in the ceramide moiety are a variable mixture of saturated and monounsaturated homologs from C ^ to C 2 , and the α-hydroxy derivatives of these fatty acids. Lignoceric (24:uf and nervonic (24:1, Δ ) acids are generally major components, along with substantial amounts of palmitic (16:0), stearic (18:0), arachidic (20:0), behenic (22:0) and tricosanoic (23:0) acids. In some cases the α - h y d r o x y fatty acids are the dominant species. Comparisons of the fatty acid composition of a wide variety of glycosphingolipids have been reported
01).
In the glycosphingolipids, ceramide is attached to a mono- or oligosaccharide by a glycosidic linkage with the primary hydroxyl group of the long-chain base. Although it has only been proved conclusively in a few instances by nmr spectroscopy or chemical synthesis, it is assumed that the glycosidic linkage between ceramide and the carbohydrate moiety
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
49
has the 3 configuration, and that the sugar residues exist in the pyranose ring form. An example is shown in Figure 1, galactosyl-(gl->-4)-glucosyl31+D-ceramide (lactosylceramide). A class of phosphoglycosphingolipids occurs in higher plants and fungi; these structures consist of carbohydrate groups with an inositol residue linked to ceramide through a phosphodiester bridge. A substance of this type from corn was proposed to be GlcNal^4GlcUAal^6inositol(2^1aMan)-l-0-phosphorylceramide (169). A related compound from tobacco leaves (170) has been shown to be GlcNAcal+4GlcUAal^2myo-inositol-l-Q-phosphorylceramide (171). In the glycoglycerolipids studied thus far, the carbohydrate residues are attached to C-3 of sn-glycerol by either a- or 3-glycosidic linkages. Three important types of glycoglycerolipids are 3-3-galactosyl-l,2-diacylsn-glycerol (12), 3'-sulfo-3-3-galactosyl-l-alkyl-2-acyl-sn-glycerol (seminolipid) (13,14), and a sulfotriglycosyldiglyceride of human gastric secretions, 3-ot-(6'-sulfoglucosyl-(a l+6)-glucosyl-(al+6)-glucosyl)-l-alkyl2-acyl-sn-glycerol (15). Structural variatio glycosphingolipids is so diverse that a special nomenclature has become necessary in referring to these substances. A semisystematic nomencla ture has been recommended by the IUPAC-IUB Commission on Biochemi cal Nomenclature (^6) and has been reviewed in some detail (6,17,18). Compounds containing a single monosaccharide unit were historically called cerebrosides. Now they are identified by the sugar they contain; examples are galactosylceramide (GalCer) and glucosylceramide (GlcCer). Similarly, the diglycosylceramide containing a lactose unit is called lactosylceramide (LacCer). More complex glycosphingolipids are given names that identify a particular portion of the carbohydrate moiety, indicating the family to which it belongs and the number of glycose units in the chain. Prefixes (given below) for the various families imply a specific structure, including the sequential arrangement of the glycose units as well as the positions and anomeric configuration of the glycosidic linkages. Thus, the glycosphingolipid of the globo type that contains all four sugar residues is referred to as globotetraglycosylceramide and is abbreviated GbOse^Cer. Similarly, the glycosphingolipid of the ganglio type with three sugar units is called gangliotriglycosylceramide (GgOse^Cer); this substance is G a l NAc3l->4Gal31^4GlcCer. Substituents that are not part of the root oligosaccharide are given at the beginning of the name, using a Roman numeral to indicate the monosaccharide unit (counting from ceramide) on which the substituent is located and a superscript Arabic numeral to indicate the position of the glycosidic linkage. According to this system, Forssman hapten (Gal Ν Ac al-* 3GalNAc31+3Galal+4Gal3 l+4GlcCer) should he called IV -a-N-acetylgalactosaminyl-globotetraglycosylceramide (I\raGalNAc-GbOse^Cer). Methods of Analysis of Glycolipids Determination of the complete chemical structure of a glycolipid usually requires a combination of several techniques. Some of the methods can be used with nanomolar amounts of sample, whereas others
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
50
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 1. Lactosylceramide (LacCer), galactopyranosyl-(βl -» 4)-glucopyranosyl—(1 -» r)-ceramide
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Prefix
Abbreviation
lacto lactoneo muco gala globo globoiso ganglio
Lc Lcn Mc Ga Gb Gbi Gg
Structure
and Metabolism
51
Structure GaKg l+3)GlcNAc(e l+3)Gal(B l-*4)Glc Gal(Bl+4)GlcNAc(ei+3)Gaiei+4)Glc Gal( β 1 +3)Gal( β 1 +4)Gal( β 1+ 4)Glc GalNAc( l+3)Gal( l+4)Gal(al+4)Gal* GalNAc(ei+3)Gal(al-*4)Gal(31+4)Glc GalNAc( Bl+3)Gal(al+3)Gal(6 l-*4)Glc Gal( β ΐ +3)GalNAc( ei+4)Gal(6 l+4)Glc
^Substances more complex than Galal->-4GalCer in this family have not so far been isolated from biological material. require about one micromole; carbon-13 nmr spectroscopy is applicable at present only if a large amount (ca. 150 mg) of sample is available. Most of the structural studies chromatographic separation of the glycolipid (column and thin-layer chromatography), followed by several commonly used analytical proce dures: fatty acid, long-chain base and carbohydrate composition by gasliquid chromatography after acid-catalyzed methanolysis; stepwise degra dation of the carbohydrate moiety with specific glycosidases; and analysis of the partially methylated alditol acetates derived from permethylated glycolipid. Frequent use of mass spectrometry has been made in recent studies to validate the structures of the partially methylated alditol acetates and to determine some structural features of the intact permethylated glycolipid. Some of the newest techniques are reviewed briefly in the following section. Excellent separations of glycolipids according to fatty acid differ ences have been obtained by high pressure liquid chromatography of perbenzoylated or O-acetyl-N-p-nitrobenzoyl derivatives (19-21), requir ing only nanomolar amounts of samples for detection at 254 nm with a suitable flow-through detector. Mixtures of natural glycolipids from GlcCer to GbOse. Cer can be separated by chromatography on Sorbax Sil (22). Though the technique appears to be ideally suited for the separation of substances with different molecular weights, sufficient studies have not been made to determine whether isomeric glycolipids (different sugar arrangement, different positions and/or configuration of glycosidic link ages, different epimeric components) can be separated effectively. An alternative chromatographic approach involves the careful analysis by thin-layer chromatography of the effluent material from a column. This technique appears to be most effective when use is made of an ion exchange material such as DEAE-cellulose or DEAE-Sephadex and when a very large number of samples from the column are spotted adjacent to each other on the thin-layer plates. The resulting map looks like a two-dimensional chromatogram with excellent resolution of components that would overlap if analyzed together on the plate. This procedure has been exploited in the study of blood group glycolipids from human erythrocytes (23) and for analyses of the ganglioside composition of brain (24,25). Additional fractionation of gangliosides has been obtained
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
52
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
on a preparative scale by DEAE-Sephadex column chromatography and subsequent separation on a new spherical silica, called Iatrobeads (26-29). Analysis of carbohydrate composition by gas-liquid chromatography is common practice. Trimethylsilyl derivatives of methyl glycosides (obtained by acid-catalyzed methanolysis) or alditol acetates (obtained by acetolysis and hydrolysis) can be used with suitable internal standards to obtain molar ratios of the sugar components with about 100 nanomoles of sample ( 3 0 - 3 2 ) · These conditions cannot be used to distinguish between N acetyl and N-glycolyl derivatives of neuraminic acid in the intact glycolipids. Mild acid-catalyzed methanolysis (0.05 Ν methanolic HC1 at 8 0 ° for 1 hr) has been used instead, giving high yields of the β - m e t h y l ketosides of N-acetyl and N-glycolylneuraminic acid, the trimethylsilyl derivatives of which separate readily by gas chromatography (33). The sequential arrangement of the sugar residues in complex glycolipids can be deduced enzymatically or by mild acid hydrolysis or methanolysis. The enzymatic procedure involves the stepwise hydrolysis of the glycolipid with specifi evidence for the nature of the terminal glycose residue at each step as well as the anomeric configuration of the glycosidic linkage (34). It is customary to first remove sialic residues from gangliosides by 1 M formic acid hydrolysis for 1 hr at 1 0 0 ° (35), a procedure which also liberates fucose (36). Negative results in the enzymatic analysis need to be interpreted with some care since they might be caused by improper detergent in the reaction mixture or a glycosidase specificity that gives poor K m or maximal velocity values with a particular structure (34). It is important to realize that the procedure requires a set of highly pure glycosidases. The other method involves partial methanolysis with 0.3 M HC1 in chloroform/methanol at 6 0 ° for 40 min; it has been used effectively for analyses of a heptaglycosylceramide (37) and various blood group Α - a c t i v e glycolipids from gastric mucosa (36). Two methods make use of permethylation for the analysis of glycolipids and can be used together to obtain information about the sequential arrangement of carbohydrate residues and the positions of glycosidic linkages. Permethylation is generally carried out in dimethylsulfoxide with a mixture of sodium dimethylsulfinylcarbanion and methyl iodide (38), a mild procedure (room temperature) which gives high yields of completely methylated product. Precautions have to be taken to exclude plasticizers from the starting materials, particularly dimethylsulfoxide. Mass spectra of the permethylated intact glycolipid reveal several kinds of structural detail, including the composition of the ceramide moiety, the nature of the terminal glycose unit, the total number of glycose units and, to some extent, the sequence of sugars (3945). Spectra of the product obtained by reduction of the N-methyl amide linkage with lithium aluminum hydride are somewhat superior as ions at high mass are stabilized and the volatility of the derivative increases to some extent (39-45). Useful information results from the cleavage reactions (at glycosidic linkages) that result from random charge localization on ring oxygen atoms. Unfortunately these ions are obscured to some extent and often represent weak contributions to the total mass spectrum, due to the presence of the ceramide moiety.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
53
An alternative approach involves conversion of the glycolipid to an oligosaccharide by ozonolysis (46) or treatment with osmium tetroxide (47), both of which attack at the double bond of the sphingosine moiety, yielding a product that is readily hydrolyzed to the free oligosaccharide. The permethylated oligosaccharide has excellent mass spectral character istics, as illustrated in the case of lacto-N-tetraose and difucohexaose I in Figures 2 and 3. The molecular ion was not observed in either case upon electron impact ionization but may be present in chemical ionization mass spectra. The ion at highest mass in the case of the lacto-N-tetraose derivative is at m/e 858; it results from the loss of a C-6 substituent (· CH^OMe) from whichever ring bears the charge (on oxygen) in the molecular ion. Three ions (below) of approximately equal intensity, at m/e 219, 464 and 668 in Figure 2, are predicted for the sequence hexoseN-acetylhexosamine-hexose-R by arithmetic calculations; they result from the cleavage of different glycosidic linkages, with charge retention on the ring oxygen and loss of the remainder of the chain as a free radical.
Ν ME AC Further loss of methanol from J^and a disaccharide from 3 both give m/e 187 (4), whereas loss of the terminal hexose residue from 2^ gives an intense ion at m/e 228 (5). The presence of m/e 464 (a hexosaminecontaining disaccharide moiety) and the absence of m/e 260 (a terminal N acetylhexosamine unit) are strong evidence for the hexose-hexosamine
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
71
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LACTO-N-TETRAOSE
464 \
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1
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Figure 2. Mass spectrum of permethyhted hcto-N-tetraose, obtained by electron impact ionization at 70 eV with a Varian MAT CH-5 double-focusing mass spectrometer (direct probe)
600
200
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SWEELEY ET AL.
55
Glycolipid Structure and Metabolism
tUC
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In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
56
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
arrangement. The presence of a strong fragment ion at m/e 228 is good evidence for a 1,3 linkage of the terminal hexose unit to the hexosamine, since this product is stabilized by the pair of electrons on the nitrogen. The product from loss of terminal hexose in a 1,4 linked disaccharide would not be similarly stabilized and would likely lose methanol to give an intense ion at m/e 196. In fact, this is the case with the doubly substituted hexosamine moiety in the difucohexaose (Fig. 3); further conversion of the ion at m/e 812 to m/e 606 by loss of the C-4 substituent results in a very weak ion, compared with that obtained by loss of the C-3 substituent from m/e 812 to give m/e 402. The ion at m/e 196, for loss of both of C-3 and C-4 substituents from m/e 812, is strong, as expected on the basis of these arguments. More precise information about the positions of glycosidic linkages can be obtained by the analysis of the monosaccharides liberated from the permethylated glycolipids by acid-catalyzed methanolysis (48) or by hydrolysis and reduction to partially methylated alditols (49-51). In either case free hydroxyl groups derivatives are analyzed b metry (50,52-54). A large number of model compounds have been analyzed and their retention behavior and mass spectral characteristics reported (55). Tfie mass spectra are similar on casual inspection but generally have at least one ion that is uniquely derived from a particular structure. Epimeric structures are differentiated on the basis of their retention behavior rather than their mass spectra. Use of this technique to distinguish between terminal, 1,3- and 1,4substituted N-acetylhexosamines is illustrated in Figures 4-6. Similarities in the mass spectra result from cleavage between C-2 and C-3 in all three cases to give an intense ion at m/e 158, which loses ketene to give another intense ion at m/e 116. As a rule strong ions are formed when cleavage can occur between two carbons substituted with O C H ^ groups, while virtually no ionization occurs by cleavage between two carbons bearing acetoxy groups. The three kinds of hexosamine are most readily distinguished by the presence of m/e 202 and 205 in the case of the product from terminal G a l N A c , m/e 233 from 1,4-linked G a l N A c and m/e 318, which is strong only in the product of 1,3-linked G a l N A c . Mass chromatography is a powerful tool that can be used to advantage for the analysis of the partially methylated alditol acetates from permethylated glycolipids, using a computerized gas chromatography-mass spectrometry system. Quick and accurate analysis of the products can be obtained without careful inspection of the total mass spectra. For example, Forssman pentaglycosylceramide (GalNAcal-*3GalNAcBl-*3Galal+4Gal31+4GlcCer) would be converted by permethylation analysis to equimolar amounts of N-methyl-N-acetyl-l,5-di-0-acetyl3,4,6-tri-O-methylgalactosaminitol from the terminal G a l N A c and N methyl-N-acetyl-1,3,5-tri-0-acetyl-4,6-di-0-methylgalactosaminitol from the internal G a l N A c residue. Mass chromatograms of m/e 205, 233 and 318 (Fig. 7) clearly show that the terminal and 1,3-substituted G a l N A c products are present in the mixture and that the 1,4-substituted G a l N A c product is not. Previous discussion of this technique by Laine (private communication, ref. 171 and Abstracts, Society for Complex Carbohydrates, New Orleans, October 8, 1976) suggests that it might be used with
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
57
Glycolipid Structure and Metabolism CH2OCOCH3 158
145
-
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ÇN^COCH-.
"205
202
205/
H
5
CH.O CH i _ L C H 3 0 CH
161
HCOCOCFL
I
3
CH2OCH3 Figure 4.
Mass spectrum of
N-methyl-N-acetyU3,4,6-tri-0-methyl-l,5-di-0-acetylgalactosamintol
43
L
Λ. 158 /
116
J
MLL
Α..
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-·
·
-
a J i a l e
-
·
CH.COO CH
I
3
H C O C O C H 3 I CH2OCH3
Figure 5.
Mass spectrum of N-methyl-N-acetyl-3fi-di-0-methyl-14,5-tri-0acetylgalactosaminitol CH2OCOCH3 43
•
Τ.,.,,.,Ιμ.,.Α
I ~ "cTÇ
116
158
y!
H
" "
318
9 ^cocH3 N
CH.COOCH 3
I
CH30-CH
H C O C O C H 3 I
CH2OCH3 Figure 6.
Mass spectrum of
N-methyl-N-acetyl^-di-O-methyl-l^^-tri-O-acetylgalâctosaminitol
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 7. Mass chromatography of m/e 205, 233, 272, and 318 along with total ion intensity (Til) for gas chromatography—mass spectrometry analysis of partially methylated alditol acetates from Forssman pentaglycosylceramide. Ion peaks of m/e 272 and 318 at scan 176 are specific for 1,3-linked GalNAc (see Figure 6), and the peak of m/e 205 at scan 149 is specific for a terminal GalNAc (see Figure 4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
59
nanomolar amounts of glycolipid. Carbon-13 nuclear magnetic resonance spectroscopy offers some unique capabilities in the structural analysis of glycolipids, not the least of which may be the possibility to analyze interactions at the cell surface. Large are required to obtain a natural abundance Fouriertransform C nmr spectrum, using a high field instrument to resolve the complex of resonances for ring carbons not involved in glycosidic bonds or otherwise derivatized. Assignments have recently been made by Sillerud et^l. (56) for the resonances of every carbon nucleus of G . , . ganglioside JÛ α - Ν - a c e t y l n e u r a m i n o s y l - g a n g l i o t e t r a g l y c o s y l c e r a m i d e ) , Based on care ful comparisons of the spectrum of this ganglioside with those of neuraminosyllactose, ceramide and several related glycolipids. We have used the assignments proposed by Sillerud et al. (56) for ^ ganglioside to analyze the C nmr spectrum of a complex neutral glycosphingolipid, globotetraglycosylceramide, and present some of the assignments in Figure 8. A higher resolution spectrum and data for a few model compounds are necessary fo In the tetrasaccharide moiety the glycosidic carbons resonate in the range typical of α and β glycosidic linkages at 100 to 106 ppm. The hydroxymethyl carbons appear at 61 to 63 ppm, the sugar ring carbons of a glycone linkage appear at 79 to 83 ppm and the ring sugar carbons not involved in inter-sugar linkages fall in the 72 to 77 ppm region, with the exception of the C-2 of N-acetyl-D-galactosamine and C-4 of the galactosyl moieties. The ceramide moiety gives a set of resonances that resemble very closely those observed for ganglioside G . . . micelles (56). The alkyl chain shows a large and broad peak for the bulk methylene carbons and sharper peaks for the terminal methyl and neighboring meth ylene carbons in the region of 14 to 40 ppm. The rest of the carbons show resonances typical of the carbonyl group (175 ppm), of a carbon-carbon double bond (132 ppm), of a hydroxymethyl carbon linked to a glycosidic oxygen (69 ppm), of a secondary alcohol (72 ppm), and of a nitrogen-linked methinyl carbon (55 ppm).
s a m e p s l
Glycosphingolipid Biosynthesis The biosynthesis of glycosphingolipids should begin with studies dealing with the synthesis of ceramide (57-59). However, from a biological standpoint, it is the composition and sequence of the glycose units that is the most interesting and most thoroughly investigated. A number of investigators have studied the biosynthesis of gluco-and galactosylceramide (60-64) and to date it is still unclear whether these compounds are made in vivo by conversion of sphingosine to psychosine and then to cerebroside, or by acylation of sphingosine to yield ceramide, from which cerebroside is derived. However, the synthesis of lactosylcer amide from UDP-galactose and glucosylceramide has been established in embryonic chicken brain by Basu et al. (64) and confirmed by Hauser and coworkers (65) in rat spleen. It should be noted here that it was primarily the finding that embryonic chicken brain homogenates are rich in various glycosyltransferases and that detergents (such as Triton X-100) greatly facilitated their measurement (66) that gave the means and impetus to
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 6
PPM
80
Figure 8. Carbon-13 NMR spectrum of globotetraglucosylceramide from porcine erythrocytes. The 15.08 MHz, proton-decoupled spectrum was obtained in a Bruker WP-60 at 35°C, using 100 mg sample in 1.5 mL pyridine-d (TMS internal standard). Transients (25,000) were accumulated at a sweep width of 3000 Hz and a 45°C pulse (8 psec). (Sweeley, Barker, and Nunez, manuscript in preparation)
100
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
61
further study glycosphingolipid biosynthesis. Lactosylceramide (LacCer) appears to be the precursor for the various classes of more complex glycosphingolipids (see Figure 9). Glycosylation of this molecule by a galactosyltransferase (65) gives globotriglycosylceramide, the precursor of globotetraglycosylceramide and globopentaglycosylceramide (Forssman hapten). Sialylation of LacCer by sialyltransferase gives II - α - Ν - a c e t y l n e u r a m i n o s y l lactosylceramide (66-68) and N-acetylgalactosaminyltransferase converts LacCer to gangliotriglycosylceramide (69), a precursor of the gangliosides. Finally, the addition of N-acetylglucosamine to LacCer by the appropriate transferase gives lactoneotriglycosylceramide, and subsequent reactions lead to the blood group-active glycosphingolipids (70). As mentioned above, studies by Basu, Steigerwald, Kaufman and Roseman (71-75) historically were the first to be reported on the in vitro biosynthesis of glycosphingolipids, in particular the gangliosides. From their work the pathway in Figure 9 was proposed. Later this pathway was corroborated in frog brai neuroblastoma clones (80). Even further support for the ganglioside pathway in Figure 9 was obtained using an in vivo strategy consisting of incubating cultured murine neuroblastoma cells with radiolabeled precur sors, followed by isolation of the glycolipid products (81). The synthesis of globotriglycosylceramide, the precursor of globote traglycosylceramide, has been established in rat spleen (65). Heat inactivation patterns and inhibition by different glycosphingolipids showed that this galactosyltransferase was different from that which catalyzes the transfer of UDP-galactose to glucosylceramide. Chien et al. (82) demonstrated a β - Ν - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e that would catalyze the synthesis of globotetraglycosylceramide from globotriglycosylcer amide and U D P - G a l N A c . More recently Kijimoto et al. (70) and Ishibashi et al. (83) have reported an α - Ν - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e activity in guinea pig tissues that can convert globotetraglycosylceramide to globopentaglycosylceramide (Forssman hapten) by addition of N-acetylgalactosamine via U D P - G a l N A c . Yeung et al (84) demonstrated the synthesis in vitro of both globoside and Forssman hapten in a clone of cultured adrenal tumor (Y-l) cells. Dawson and Sweeley (85) concluded that the synthesis of globotetraglycosylceramide of porcine erythrocytes occurrs in bone marrow cells. Studies on the biosynthesis of the blood group-active glycosphingo lipids have also been made (see Figure 10). Basu and coworkers have reported the synthesis of lactoneotetraglycosylceramide from lactotriglycosylceramide (the core blood group structure) and a blood group B-active pentaglycosylceramide from lactoneotetraglycosylceramide by 3-galactosyltransf erase (86) and an α - g a l a c t o s y l t r a n s f e r a s e (87) isolated from rabbit bone marrow cells, respectively. They also reported the presence of galactosyltransferase activities involved in the synthesis of this blood group B-active glycolipid in cultured mouse neuroblastoma cells (80). GDP-fucose:glycolipid fucosyltransferase activities related to blood group structures have also recently been demonstrated. The biosynthesis in vitro of blood group H . Gal(2 -*4a Fuc) gl-*~4GlcNAc6 1-*· 3Gal e i + 4 G l c C e r {88^9j~and human B-type A Gala l + 3 G a l ( 2 « - l a F u c ) β1+ 4GlcNAcBl+ 3Galgl+^GlcCer (90) glycosphingolipids have been described in a Golgi-enriched
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Galactosyltransferose
Figure 9.
N-Acetylgalactosaminyltransferase
Sialyltransferase
4Glctfl^lCei
N-Acetylgalactosaminyl transferase
CMP-NeuAc^
Sialyltransferase
PAPS
UDP-Gal NAc^j
G A N G L I O
T Y P E
Sialyltransferase
3 Ga I Ν A c £ l - 4 G a ! ( 3 - 2rXNeu Ac )β 1 - 4 GI cB I - 1
Galactosy I transferase
T Y P E
glycosphingolipids
NeuAca2~3Gaim-3GalNAcâl-4Gal(3~20NeuAc)£1^4Glci31-
Ga l â 1 -
UDP-Gal-
Ce r
N- Acetyl galactosaminyl transferase
3
» 3-S0 -GalaH4Gal/?WCer
GalNAcfll^4Gal(3-2aNeuAc)51—4Glc£l~lCer
G A L A
Sulfo^onsferose
Gal^l-3GalNAc01^4Gal(3^2CrNeuAc8^2aNeuAclffl-4Glc81-lCer
Galactosyltransferose
Ga INAc#l - 4 G a l ( 3 - 2KNeuAc 8~20CNeuAc)/31- 4 G l c â l - ICer
UDP-GalNAc-
1
Ga1al-4Gol/a-lCer
NeuAciC2 — 8 N e u A c # 2 — 3 G a l # l - 4 G l c , S l — ICer
—
Sulfotransferase
Pathways for the biosynthesis of sulfo-, neutral (Forssman hapten), and acidic (gangliosides)
T Y P E
a 1 - 4 Gal/31 - 4 G l c / S l - 1 Cer
N
G L O B O
G a l N A c a l - 3 G a l N A c β\-3Gal
UDP-GalNAc
CMP-NeuAc
NeuAcOC2^3Galâl
CMP - NeuAc -
Sialyltransferase
Galactosyltransferose
Gal/01-4Glc/Sl-lCer
UDP-Gal
GlC/Gl - I C e r
• Gal/91-lCer UDP-GalJ 1 Galactosyltransferose
NeuAcC* 2-lGQ\fi\*~5Q>Q\mce\ - 4 G a l ( 3 - 2 C t N e u A c 8 ~ 2 û C N e u A c ) / 3 l - 4 G l c / ? W C e r
N-Acetylgalactosaminyltronsferase
GalNAc/Sl-3Galai-4Gal/Sl-4Glc/3l-lCer
UDP-GalNAc >
Galal-4Gal/31~4Glc/3l-lCe-
w,N
~ j
UDP-Gal Gclactosyltransferase
UDP-GlcΊ Glucosyltransferase
Ceramide
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. jGalactosyltransferose
Fucosyltransferase UDP-Gal
JGalactosyltransferose
Figure 10.
TYPE
N j
Galoctosyltransf erase
LACTO
TYPE
Pathways for the biosynthesis of blood group-related
LACTONEO
Blood Group B - l
l
Fucosyltransferase
Blood Group Β
Blood Group A
u
UC
N
Gal(2-la F )/S 1 -3GlcNAc β l-3Gal 3 l-4Glc/31-lCer
GDP-Fuc
Gakxl-3Gal(2-laF c)/8l-4GlcNAc/91-3Gal/?l-4Glc/3l-lCer
|N-Acetylgolactosaminyltransferase
Gal(2MaFuc)tfl-4acNAcfll-3Gol<3l-4Glc/3l-«lCer
GDP-Fuc Ν
jGaloctosyltransf erase Galtfl-3GlcNActfl-3Gal/3l-4Glc/3l--lCer Galoctosyltransferase
• Galcri-3GaU?l-3GlcNAc51-3Gal/31— 4 G l c â l — lCer
glycosphingolipids
Gala 1 -3Gal(2-la Fuc) β l-3GlcN Ac/? 1 -3Gal β 1 -4Glc β 1 -1 Cer
- GlcNAc9l~3Gal#i-4Glc/3HCer
Gal(3l-4GlcNAc/9l-3Gol/Sl-4Glc/91-lCer
Fucosyl transferase
UDP-GlcNAc.l . . , \N-Acetylglucosaminyltransf erase
GalNAcal-3Gal(2-laFuc)/Sl-4GlcNAc/i3l-3Gol Sl*4GlQSl-lCer
UDP-GalNAc χ
Galactosyl transferase
GlcNAc(0CFuc)-flt-3Gol^l- 4Glc51-tCer -
GDP^Fuc
GalSl-4Glc£l-lCer
S" ο
Ci
a,
"S
Ci Ο
> t-1
w w
i
GO
64
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
fraction isolated from bovine spleen. Pacuzka and Koscielak (91) also demonstrated the synthesis of these glycolipids with enzymes from human serum. Moreover, Presper et al (92) found a fucosyltransferase in human (IMR-32) neuroblastoma cells that is involved in the synthesis of types H - l and B - l glycolipids as well as a tetraglycosylceramide precursor of a novel L e a blood group glycosphingolipid. Many other glycosphingolipids have been purified and structurally characterized but their biosynthesis in tissues or cultured cells have not yet been determined. Two particularly interesting groups are the branched blood group-active glycolipids such as those reported by Watanabe et al. (93) and Slomiany and Slomiany (94) and the "macroglycolipids" such as those characterized by Gardas and Koscielak (95). The above data establish that glycosphingolipids are synthesized by the stepwise addition of activated sugars directly to the appropriate glycolipid acceptor. It will be interesting to see if this holds true for the "macro-glycolipids" as well, since they appear to contain a number of repeating units. Perhaps intermediat those described in glycoprotein biosynthesis. In conclusion, increasing numbers of reports are appearing in which glycolipid biosynthetic pathways are demonstrated in various cell culture systems. Using such model systems in concert with the characterization of endogenous and cell surface glycoconjugates should be very helpful in furthering our understanding of the role of glycosphingolipids in cellular processes and tumorigenesis. Acceptor specificity of
glycosyltransferases
The synthesis of the carbohydrate moiety of glycosphingolipids apparently takes place in a sequential manner and is catalyzed by glycosyltransferases with specificity for both nucleotide sugar donor and glycolipid acceptor. Until recently (97), purified glycosyltransferases have not been available to test for acceptor specificity with glycolipid substrates. Therefore, judgments have been made on the basis of indirect evidence (Table I) Hildebrand and Hauser (65) compared the heat lability and metal ion requirements of galactosyltransferases from rat spleen. A galactosyl transferase utilizing glucosylceramide as an acceptor molecule was inactivated by heat treatment ( 5 0 ° C for 5 minutes), while lactosylceramide galactosyltransferase activity was only slightly affected (20% reduction). Although both transferase activities were stimulated by manganese, only glucosylceramide galactosyltransferase was enhanced by magnesium. A third difference between the two transferase activities was their response to the addition of various sphingolipids. Lactosylceramide synthesis was inhibited by sphingosine, galactosylsphingosine, lactosylsphingosine and ceramide, whereas globotriglycosylceramide synthesis was inhibited by lactosylsphingosine, ceramide and galactosylceramide. Substrate competition experiments have shown that rabbit bone marrow contains two galactosyltransferase activities, 3-galactosyltransferase synthesizing lactotetraglycosylceramide and an a-galactosyltransferase synthesizing lactopentaglycosylceramide (820. When comparisons
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. P a c u s z k a and K o s c i e l a k (1976) K a u f m a n et a[. (1963) F i s h m a n et a l . (1972) S c h w y z e r and H i l l (1977)
Heat l a b i l i t y Viral t r a n s f o r m a t i o n Pure enzyme
LacCer,II3aNeuAc-GgOse^Cer
LacCer,II3aNeuAc-GgOse^Cer
Bloodj^roup g l y c o l i p i d s , A , Le , H - m e g a and H
N e u A c - t r a n s f erase
N e u A c - t r a n s f erase
GalNAc-transferase
Fuc-transferase
Gene expression
LcOse3Cer,LcOse^Cer
G a l - t r a n s f erase
LcnOse^Cer asialoganglioside
GlcCer,LacCer
G a l - t r a n s f erase
Basu and Basu (1973)
Reference
Substrate c o m p e t i t i o n
Evidence for Acceptor Specificity
H i l d e b r a n d and Hauser (1969)
Acceptors
Glycosyltransferases
1. M e t a l ion 2. Heat l a b i l i t y 3. Inhibition by sphingolipids
Enzymes
A c c e p t o r S p e c i f i c i t y of
TABLE I
66
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
were made of C-galactose incorporation (from UDP-galactose) in the presence of each acceptor individually and when both acceptors were present together, an additive relationship was found. The authors therefore concluded that two galactosyltransferases were present. Fucosyltransferase activities have been measured in human serum, with lactoneotetraglycosylceramide and "asialoganglioside" as acceptors (91). Serum of A B O and Lewis blood group contained fucosyltransferase activity with both acceptors, while serum preparations from two Bombay blood donors (O, ) lacked asialogangliofucosyltransferase activity. Thus, the two fucosyltransferase activities appear to be products of different genes. Reports by Kaufman et al. (73) and Fishman et al. (96) demonstrated that glycosyltransferases with glycolipid products of the same anomerity may be different proteins. They differentiated similar sialyltransferase activities (with lactosylceramide and N-acetylneuraminosylgangliotetraglycosylceramide as acceptors) on the basis of in vitro (heat lability) and in vivo (viral transformation lactosylceramideisialyltransferas II -a-N-acetylneuraminosylgangliotetraglycosylceramide:sialyltransferase. Fishman et al. (96) demonstrated that Swiss 3T3 cells contain these same sialyltransferase activities. Following transformation by SV-40 virus, N acetylneuraminylgangliotetraglycosylceramide sialyltransferase was re duced, whereas lactosylceramideisialyltransferase activity did not change. In all of the cases cited above, enzyme activities were measured in homogenates or membrane preparations. Recently Schwyzer and Hill (97) reported on the acceptor specificity of a homogeneous preparation of N acetylgalactosaminyltransferase from porcine submaxillary glands. The enzyme specifically transferred N-acetylgalactosamine from U D P - G a l NAc to substrates (both glycoprotein and glycolipid) with a blood group H structure. Fucod+2Gal Bl->4GlcNAc β1-> 3Gal 61->4Glc Cer and H-megg glycolipid were both acceptors for the enzyme, while A and Le glycolipids did not serve as substrates. The studies discussed above certainly suggest that glycosyltransfer ases display a specificity for glycolipid acceptors; however, some references can be cited which argue for nonspecificity. Chien et al. (82) found a single N-acetylgalactosaminyltransferase from embryonic chicken br^in that could utilize lactosylceramide, globotriglycosylceramide and II -N-acetylneuraminosyl-lactosylceramide as glycolipid acceptors, based on the results of substrate competition experiments. The same workers (88) have used a similar experimental approach to demonstrate multiple acceptor recognition with a fucosyltransferase from bovine spleen. Although several studies have dealt with the question of acceptor specificity, little is known concerning the possible role of protein modifiers (e.g., " A protein" in lactose synthase) and chemical alteration (i.e., phosphorylation, adenylation) in glycosyltransferase reactions. In the reports discussed above, the acceptor specificity (or lack of) may be a consequence of the presence or absence of such modifications or alterations. These ideas will be discussed further in the section dealing with the regulation of glycosyltransferases.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
67
Glycosyltransferase localization Roseman has proposed that glycosyltransferases on the surface of mammalian plasma membranes may be involved in intercellular glycosylation, i.e., glycosylation of surface glycoproteins and glycolipids of one cell by glycosyltransferases on the surface of an adjacent cell, and that enzyme-substrate complexes of these enzymes could be the key mechanism governing intercellular adhesion and thus, possibly, tumorigenesis (75). Since that time a large body of data has emerged, both in support of this idea (see review by Shur and Roth (HO)) as well as against it (see review by Keenan and Morre (111)). In order to establish the presence of cell surface glycosyltransferases, a number of criteria must be fulfilled. The purity of isolated membranes must be very carefully established by both membrane marker enzymes and electron microscopy. When using whole cells, one must control for possible hydrolysis of sugar nucleotides and transport of free sugars into the cell, and enzym carefully monitored. Further, glycosyltransferase assays must be optimized under all conditions examined. A number of investigators arguing for the presence of cell surface glycosyltransferases have controlled for sugar nucleotide breakdown (112-114). Others have controlled for intracellular glycosyltransferase "leakage" (112,115-117). Upon closer inspection of the experimental design of these various studies, it can be seen that none has included all of the appropriate controls, as discussed above. Keenan and Morre and their coworkers have presented the strongest evidence against cell surface glycosyltransferases. Figure 11 depicts the membrane flow hypothesis put forth by Morre (118,119). In essence, membrane biosynthesis includes the actual transfer of membranous material from one subcellular site to another, finally reaching (via fusion) the cell surface. Endoplasmic reticulum membranes are transferred to the Golgi-apparatus where they undergo modification to become plasma membrane-like. Completed membrane material is then incorporated into secretory vesicles which move to and fuse with the plasma membrane, discharging their intravesicular contents into the extracellular milieu. Morre goes on to postulate such a route for cell surface glycolipids and glycoproteins. Initial data to support the ideas above came from electron microscopic autoradiography (120) where it was shown that glycosylation of glycoproteins takes place in the Golgi-apparatus. Studies with highly purified and well characterized membrane fractions shortly followed. High specific activities for several glycoprotein (121) and glycolipid (122) glycosyltransferases in Golgi-apparatus preparations were established. More recently, lactose synthase (123) and glycosyltransferases for ganglioside biosynthesis (124) have been demonstrated in mammary gland and liver Golgi, respectively. Glycosyltransferase activities concentrated in the Golgi-apparatus of small intestines (125), pancreas (126), thyroid (127), and many other tissues have also been reported. The strength of this work relies on (1) the careful demonstration of purified membranes, (2) the significantly higher specific activity of glycosyltransferases in
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
j
VACUOLE
NUCLEUS Figure 11. Synthesis of cell surface glycoconjugates according to the "membrane-flow" hypothesis
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
69
Golgi-membranes compared to plasma membranes or homogenatess and (3) autoradiographic localization of radiolabeled glycose moieties in Golgi and endoplasmic reticulum. To date no conclusive evidence has been given for intercellular glycosylation of lipids or proteins by surface glycosyltransferases. However, many reports have appeared which implicate cell surface glycosyltransferases in a wide varity of recognition phenomena (116,128133). In conclusion, it seems quite reasonable to assume that there are glycosyltransferases on the cell surface, based on the number and diversity of reports, even if all of the appropriate controls have not been adequately considered. However, conclusions as to their function are tentative at best. The fact that glycosylation enzymes are found in the Golgi-apparatus seems to be well established. Moreover, the idea that some glycosyltransferases appear on the cell surface via "membrane flow" and function only as membrane repair systems cannot be ruled out. Clearly, this area of researc further studies will provid complex glycoconjugates, irrespective of whether the functionally most important glycosyltransferases are on the cell surface or in the Golgiapparatus. Regulation of
glycosyltransferases
Several studies (98-101) with glycoprotein glycosyltransferases have demonstrated an in vitro effect of nucleotide phosphates. Both inhibitory and stimulatory effects have been observed. Enzymes utilizing glycolipid acceptors are also affected by adenine- and uridine-containing compounds (Table II). In a study by Ishibashi et al. (102) both positive and negative modulating effects on N-acetylgalactosaminyltransferases from guinea pig kidney were noted with uridine nucleotides. Interestingly, the a transferase which catalyzes the synthesis of globopentaglycosylceramide was markedly inhibited by U D P while the β - t r a n s f e r a s e (globotetraglycosylceramide synthesis) was activated. UDP-glucose was similar to U D P in its effects on the transferases, whereas several other uridine compounds suppressed both activities. Other information has been presented on the possible regulatory effects of substrates and products of glycosyltransfer ases and is referred to in Table II. Lactose synthase provides an interesting example of glycosyltrans ferase regulation (103). In vitro, α - l a c t a l b u m i n causes galactosyltransfer ase to become a lactose synthase. In the absence of a-lactalbumin, glucose is a very poor acceptor (Km > 1M) and the enzyme serves as a glycoprotein galactosyltransferase. The possibility that protein modifiers exist for the regulation of glycolipid synthesis can now be examined. The availability of techniques to obtain purified glycosyltransferases, along with cell systems, in which viral transformation (see section on Acceptor Specificity) appears to result in a specific loss of a glycosyltransferase, should provide the means to test such a possibility. The regulation of glycosyltransferase activity has been demonstrat ed in intact animal and cell culture systems (Table II). The level of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
70
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES TABLE II REGULATION O F G L Y C O S Y L T R A N S F E R A S E S GLYCOSYLTRANSFERASE(S)
EFFECTOR
EFFECT (+ or -)
REFERENCE
In Vivo UDP-Gal:Ceramide Galactosyltransferase
Testosterone
ω
Gray ( 197l) a
U D P - G a l : G M l Galactosyltransferase, UDP-Gal:Glucosylceramide Galactosyltransferase, UDP-Gal:Lactotriosylceramide Galactosyltransferase
dBcAMP
ω
Basu etal.(1976) b
CMP-NAN:Lactosylceramide Sialyltransferase
Butyrate
ω
Simmons et al. (1975).c M â c h e r , et al. ( 1 9 7 7 Γ
GDP-Fucose:neolactotetraosylceramide Fucosyltransferase
6-mercaptoguanosine
ω
Presper é t a l . (1978)e
In Vitro Lactose synthase
Morrison, 3.F. and Ebner K . E . (1971)8
substrate inhibition
Porcine blood group A:N-acetylgalactosaminyltransf erase
UDP Fucosyllactose
(-) (-) (-)
UDP-Gal:Glucosylceramide and Lactosylceramide Galactosyltransf erases UDP-Gal Ν A c : Tr igl ycosylcer am ide N-Acetylgalactosaminyltransf erase
Uridine UDP UDP-Glucose UDP-Galactose
UDP-GalNAc:Globoside N-Acetylgalactosaminyltransf erase
Uridine UDP UDP-Glucose UDP-Galactose
Schwyzer and Hill (1977)h
Chandrabose, K . A . , and. MacPherson, L A . (1976)1 Ishibashi et al. (1976)j
* G . M . Gray (1971) Biochem. Biophys. Acta, 239, <»9<>-500. Basu, S., Moskal, 3.R. and Gardner, D . A . , (1976) "Ganglioside Function:Biochemical and Pharmacological Implications , (Porcellati, G . , ed.) Plenum Publishing Corp., N . Y . p. *5-63. ^Simmons, 3.L., Fishman, P . H . , Freese, E., and Brady, R.O., (1975) 3. Cell Biol., 66, 414-424. d Macher, Β . Α . , Lockney, M . , Moskal, 3.R., Fung, Y - K . and Sweeley, C.C.,tl978) Exp.. Cell Res., unpublished, f Presper, K . A . , Basu, M . , and Basu, S., (1978) Proc. Nat. Acad. Sri. U.S.A., 75, 289-293. f B e l l , J.E., Beyer, T . A . and Hill, R.L., (1976) 3. Biol. Chem., 251, 3003-3013. ^Morrison, 3.F. and Ebner, K . E . , (1971) 3. Biol. Chem., 246, 3992-3998. "Chandrabose, K . A . and MacPherson, I . A . t 7 Ï 9 7 6 T B ï ô c h i m . Biophys. Acta, 429, 96-111. !Schwyzer, M.and Hill, R . L . , (1977) 3. Biol. Chem., 252, 23*6-2355. J Ishibashi, T., Atsuta, T. and Makita, Λ Τ Π 9 7 6 ) Biochim. Biophys. Acta, » 2 9 , 759-767. b
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digalactosylceramide in the kidneys of female C 3 H / H e and C57/BL mice was considerably lower than in male mice of the same strains (104). Administration of testosterone to the female mice resulted in a remarkable increase in digalactosylceramide levels. Enzymatic studies demonstrated a regulatory effect by testosterone on UDP-galactose:ceramide galactosyltransferase in these mice. Further information concern ing steroid regulation of glycosyltransferases has not been published. Basu et al. (105) assayed a number of glycosyltransferase activities in clones (NIE-115, adrenergic and NS-20, cholinergic both derived from C-1300 tumors of A/3 mice) of mouse neuroblastoma cells and found that three enzymes involved in glycolipid synthesis were stimulated by Ν , Ο dibutyryladenosine cyclic 3,,5'-monophosphate (dBcAMP) (Table II). Lactoneotetraglycosylceramide synthesis was increased 5-fold in NIE-115 cells and 2-fold in NS-20 cells. UDP-galactose:glucosylceramide galacto syltransferase activity was increased 3-fold in NS-20 cells and remained unchanged in NIE-115 cells, while the synthesis of II a-N-acetylneuraminosylgangliotetraglycosylceramid treated NS-20 cells. It is possible that d B c A M P may exert its stimulatory effect on glycosyltransferases via phosphorylation. Alternatively, these changes may reflect alterations in cell cycle distributions of the cell populations. This suggestion would be in line with findings presented by Wolf and Robbins (106) and Chatterjee et al. (107) that glycolipid synthesis is greatest during certain portions of the cell cycle (M, G j ) . Our own studies (Ref. 108 and Fig. 12) and those of Simmons et al. (109) on the effect of butyrate in cultured human cells demonstrated that glycosyltransferase levels can be adjusted within hours of drug treatment. Simmons et al. (109) originally showed that butyrate treatment of HeLa cells for 18-24 hr resulted in a 15-20 fold increase in CMP-sialic acid:lactosyltransferase activity and that this enzyme activity returned to control levels when the butyrate was removed. M â c h e r et al. (108) have studied the same enzyme in K B cells and found a similar induction. They have also demonstrated that K B cells apparently are synchronized by butyrate treatment (Fig. 13). Furthermore, release from butyrate treatment and addition of fresh medium resulted in a remarkable induction of UDP-Gal:lactosylceramide galactosyltransferase (Fig. 14). Therefore, it seems that butyrate treatment and release results in differential activation of sialyltransferase and galactosyltransferase activities in human cells. Glycosphingolipid Catabolism Virtually all the enzymes necessary for the stepwise catabolism of glycolipids have been studied in vitro and in many cases they have been partially or completely purified. These enzymes, which are exoglycosidases, have received much attention because of their involvement in the hereditary lipidoses. Recently, the potential of using enzyme replacement therapy for the treatment of these diseases has been examined. Another effort in the field has been directed toward an understanding of the kinetic properties and specificity of these enzymes. A brief discussion of the exoglycosidases involved in glycolipid metabolism is given below.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
0
4
8
f-
12
(+) Butyrate
16
20
& 4·
4
8
12
16
20)
24
(-) Butyrate
Figure 12. C-thymidine uptake after release from butyrate and double thymidine block in KB cells. Redrawn from Simmons et al., J. Cell Biol. (1975) 66,414. 14
Figure 13. The effect of butyrate on CMP-NAN:hctosyheramide sialyltransferase activity in HeLa cells. ( O — O ) Release from butyrate; (·—·) release from thymidine block.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Occurrence and inducibility Glycosidases are widely distributed in microorganisms, animals and plants. Neuraminidase, for example, was first discovered in a virus (134) and later found to occur in a variety of microorganisms, avian and mammalian tissues. The induction of specific bacterial glycosidases in the presence of the corresponding substrate is well documented. For example, α - g a l a c t o s i d a s e and β - g a l a c t o s i d a s e are not constitutive but are inducible in E . coli (135). With regard to tissue and cellular distribution, the glycosidases occur widely in organs (brain, kidney, liver and spleen), bone marrow, plasma, spermatozoa, platelets and erythrocytes. Although some glycosidases have been found in mitochondria (136), plasma membranes (137) and chloroplasts (138), the lysosome is the only organelle that contains the full complement of hydrolase activities necessary for the complete degradation of glycolipids and glycoproteins. Purification and physical propertie Virtually all glycosidases purified to date are glycoproteins; several kinds of purification procedures are available that are based on affinity methods. In re'cent years, immobilized substrate analogs have been used to facilitate the purification of several glycosidases (139). Their purification can also be accomplished by the use of immobilized plant lectins specific for certain carbohydrate units on the enzymes. Concanavalan A-Sepharose, for example, has been used for the isolation of glycosidases such as a-N-acetylgalactosaminidase (140) and the β - Ν acetylhexosaminidases. Variation in the composition of the carbohydrate moiety leads to microheterogeneity, for example molecular weight and charge, in an otherwise homogeneous glycosidase preparation. Variation in sialic acid content has often been cited as a cause of multiple isoelectric forms of these enzymes. Multiple forms of L-fucosidase , for example, have been shown (141) to be convertible to the neutral forms by neuraminidase treatment. An excellent discussion on the issue of homogeneity of glycoproteins has appeared elsewhere (142). Another problem of the multiple forms, which we wish to emphasize, has to do with substrate specificity. Often, the presence of a contaminating glycosidase cannot be readily detected unless the incubation time is sufficiently long. This is especially important to realize when glycosid ases are used for structural eludication of glycoconjugates. L i and L i , for example, have shown (34) in studies of the core glycopeptide of ovalbumin that the liberation of one of the mannosyl units was due to the presence of 0.06% β -mannosidase in an apparently homogeneous α -mannosidase preparation. Substrate specificity The substrate specificities of glycosidases are much more complica ted than their names imply. Figure 15 shows the^structures of several glycosphingolipids (globopentaglycosylceramide, II ,IV - α , α - d i - N - a c e t y l neuraminosyl-gangliotetraglycosylceramide, and fucolipid A a ) and the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
74
Figure 14. The effect of butyrate on UDPgal'Mctosylceramide a-galactosyltransferase activity in KB cells
°
2 f
4
6
- (^Butyrate
2
*
2
4
•+· Time (hrs)
6
Globopentaglycosylceramid A Β C D Ε G a l N A c ( a l +3)GalNAc(B 1+ 3)Gal(a 1-*)Gal( 81 -4)Glc( β 1 1 OCer
II-,IV--a,g-Di-N-acetylneurarriinosyl-RanRliotetraRlycosylceramide G H I Κ Galtë 1-»· 3)GalNAc( Bl+*)Gal(0 l+4)Glc(B 1* 1 OCer (a2-3)F
(a2-3) 3 NeuAc
NeuAc
Fucolipid A L Ν Ο Q R G a l N A c ( al+3)Gal(B l - ^ ) G l c N A c ( Bl+3)Gal(3 l ^ ) G l c ( B 1 + l')Cer |(al-2)M Fuc
LINKAGE A, L Β, Η, Ο C D, G , I, N , Q Ε =Κ = R F, 3 Μ, Ρ
2
(-) Butyrate
|(al->3)P Fuc
ΕΝΖΥΜΕ a-N-Acetylgalactosaminidases Β -Hexosaminidases α-Galactosidases β-Galactosidases β-Glucosidases Neuraminidases α-Fucosidases
Figure 15. Structures of three glycosphinpolipids and the exoglycosidases which catalyze the sequential hydrolysis of glyco lipid glycosidic linkages (A to R)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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corresponding exoglycosidases involved in their metabolism. Several points regarding the mode of action of these enzymes follow. Glycosidases from different sources often have different specificities. Several kinds of neuraminidase have been isolated, each of which has unique properties. Degradation of G n . begins with the removal of the sialic acid linked to the galactose aT trie non-reducing end (Figure 15, linkage F), using neuraminidase from Vibrio cholerae. The other sialic acid of G ^ j is r é s i s t e n t to this enzyme (as is the product) (143). Removal o i frie vicinal N-acetylgalactosamine unit irdm G . . . is necessary before this neuraminidase can hydrolyse the ketosidic linKage shown in (Figure 15, linkage J). It has been shown, however, that neuraminidase from Clostridium perfringens (144) can remove this sialic acid (linkage 3) from GJ^J directly; thus the specificity of a given enzyme often varies depending upon its source. While each glycosidase has an absolute specificity toward the anomeric linkage, absolut This is shown by compariso acetylhexosaminidase. α - Ν - a c e t y l g a l a c t o s a m i n i d a s e lacks specificity for the C-2 substituent as well as its configuration on the carbohydrate position at the C-2 (145,146). As shown in Figure 16, the enzyme can hydrolyze all three substrates (at different rates) which differ in the substituent and/or absolute configuration (shaded portions) at C-2. The enzyme, however, does show absolute specificity at the C-4 position. In contrast, β - Ν - a c e t y l h e x o s a m i n i d a s e has an absolute requirement for an equatorial acetamido moiety at C-2 but the configuration at C-4 is not important, as shown in Figure 17. Moreover, α - g a l a c t o s i d a s e A has been shown (147) to have an absolute requirement at both C-2 and C-4 positions and is thus distinguished from the Β form of α - g a l a c t o s i d a s e (15,16). Dean et al. (147) and Schram et al. (148) have shown that α - g a l a c t o s i d a s e Β is an o-N-acetylgalactosaminidase. Multiple forms of a given enzyme may exist which differ in their ability to hydrolyze glycolipids but not artificial water soluble substrates. Two forms of human β - g a l a c t o s i d a s e have been shown to hydrolyze the artificial substrate, 4-methylumbelliferyl- β - D - g a l a c t o p y r a n o s i d e (149,150). One of these forms ( G M .-ganglioside: β - g a l a c t o s i d a s e ) was more specific for G . , , - and asialo υ M 1 -ganglioside, while the other form (galactosylceramidase) was more specific for galactosyl- and lactosyl ceramide. Similarly, two forms of β - Ν - a c e t y l h e x o s a m i n i d a s e have been found which catalyze the hydrolysis of globotetraglycosylceramide and 4methylumbelliferyl-B-N-acetylhexosaminide. Only οτψ ( β - Ν - a c e t y l h e x o s aminidase A) of these forms was able to hydrolyze II -N-acetylneuraminosyl-gangliotriglycosylceramide. Two forms of human β - g l u c o s i d a s e have been identified. Both hydrolyze 4 - m e t h y l u m b e l l i f e r y l - β - D - g l u c o p y r a n o side but only glucocerebroside: g-glucosidase is capable of hydrolyzing glucocerebroside (151,152). This form of the enzyme appears to be lysosomal in origin.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Journal of Cell Biology
Figure 16. Three-dimensional of terminal carbohydrate residues that bind to a-N-acetylgahctosaminidase. R represents the remaining portion of the sphingolipid. Substituents that the en zyme cannot distinguish are shaded (109).
Journal of Cell Biology
Figure 17. Three-dimensional projections of ter minal carbohydrate residues which bind to β-Νacetylhexosaminidases. R represents the remaining portion of the sphingolipid. Substituents that the enzyme cannot distinguish are shaded (109).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Lipid-protein interactions Detergents are essential for the dispersion of most glycolipid substrates in glycosidase assay mixtures. In addition, it has been shown that detergents can stimulate or inhibit glycosidases directly. Presum ably, detergents mimic the effect of naturally occurring membrane amphiphilic lipids. β - G l u c o c e r e b r o s i d a s e : β - g l u c o s i d a s e , for example, has been shown to be activated by bile salts (151,153,154) such as deoxycholate, glycocholate and taurocholate; acidic phospholipids such as phosphatidylserine, phosphatidylinositol and phosphatide acid are also active (152,155). Interest ingly, the water-soluble β - g l u c o s i d a s e , which does not have activity toward lipid substrates, is inhibited by these amphiphiles. On the other hand, the β - g l u c o c e r e b r o s i d a s e : β - g l u c o s i d a s e , which hydrolyzes lipid substrates was inhibited by choline-containing lipids. Ho (156,157) has proposed a glucocerebrosidase enzyme system composed of glucocerebro sidase (factor C), a glycoprotei phospholipid. The acidic phospholipid appears to be necessary for the association of the activator and the enzyme. The activator-enzyme complex hydrolyzes artificial water-soluble substrates and G l c C e r . Sodi um taurocholate can substitute for the activator. Recently, Hechtman (158)Jias reported the isolation of an activator required for the hydrolysis of II -a-N-acetylneuraminosyl-gangliotriglycosylceramide by β - h e x o s a m i n i d a s e A . This activator does not affect the rate of hydrolysis of synthetic substrates or gangliotriglycosylceramide. Modulation of other glycosidase activities has also been reported. L i and L i (159) have isolated an activator which stimulates the hydrolysis of a number of glycolipids. However, this activator may be interacting with substrate rather than enzyme, judging from the fact that a 1:1 molar ratio of activator and substrate is required. , Synthetic lipoidal activators have also been reported (160). Figure 18 shows the structures of some synthetic fatty acyl amides which resemble fatty acyl sphingosine. The hydrolysis of galactosylceramide by β - g a l a c t o s i d a s e was stimulated by fatty acyl derivatives of 2-amino-2methyl-l-propanol, with the N-decanoyl derivative being the most active. In general, the V but not the Κ was changed. Omission of the branched methyl group resulted in inhilfftion instead of stimulation. The reports cited above suggest that protein activators stimulate the activity of glycosidases toward glycolipid substrates but do not modify the enzymatic activity toward water-soluble substrates. Our recent studies on the interaction of sodium taurocholate with a-N-acetylgalactosaminidase from canine liver (146,161) indicate that amphiphiles may stimulate the hydrolytic activities of glycosidases toward both artificial water-soluble substrates and glycolipid substrates. Low concentrations of sodium taurocholate stimulate the activity of the enzyme toward the artificial substrate Ρ Ν Ρ - α - G a l N A c . However, as the concentration of taurocholate is increased, inhibition results. A Lineweaver-Burk plot indicates that sodium taurocholate, at all concentrations, lowers the V m a x and the Κ for the hydrolysis of the artificial substrate. The percent maximal inhibition levels off at the critical micelle concentration of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES H I C — I H
Figure 18. Fatty acid amides which affect hydrolytic activity of rat brain β-galactosidase toward galactosylceramide. The N-decanoyl derivative was the most effective (160).
^
X
«
ChU
H I I C — C I I NH H I C-O I
—
6 , 8, 10, 12, 14,
OH
16
GLYCOSIDASES
φ ®
RELEASE OF G L Y C O S I D A S E S RECOGNITION OF ENZYME BY RECEPTOR
© ©
UPTAKE OF ENZYME PY P I N 0 C Y T 0 S I S FUSION OF PINOCYTIC VESICLE WITH P R I M A R Y
LYS0S0ME
Figure 19. Pictorial model of exocytosis and endocytosis of lysosomal glycosidases
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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taurocholate (7 mM). Moreover, sodium taurocholate at very low concentrations significantly quenches the intrinsic fluorescence of the enzyme. Our data (161) indicate that the enzyme activity is modulated by the interaction with the monomeric but not the micellar form of taurocholate. These studies may shed light on the mode of action of glycosidases in their natural membrane environment. Physiological significance of glycosidases Besides the well publicized lipidoses resulting from glycosidase deficiencies, attention has shifted toward the release and uptake of these enzymes from cells. An abundance of evidence indicates that tumors, studied in vivo, release large quantities of lysosomal enzymes into their environment (162-164). Presumably this release of degradative enzymes is associated with tumor invasiveness and maintainance of the neoplastic state. Other studies (dealing with I-cell disease) have shown that specific recognition molecules on lysosomal glycosidases. A diagram of these processes is depicted in Figure 19. The uptake depends upon both the carbohydrate moiety of the glycosidases (165,166) and the presence of specific receptors on the plasma membrane (167,168). Clearly, more detailed studies are needed, results of which should be significant in the design of enzyme replacement therapy. Acknowledgements The authors would like to especially acknowledge the secretarial work of Ms. Dorothy Byrne. Mr. Kenneth J. Dean and Dr. S.-S.J. Sung are also gratefully acknowledged for providing data dealing with a - N acetylgalactosaminidase and α - g a l a c t o s i d a s e . This work was supported by NIH research grants (AM 12434 and H L 17462).
Literature Cited 1. 2. 3. 4. 5. 6. 7.
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66. Basu, S., Ph.D. Thesis, Univ. of Michigan, Ann Arbor, Michigan, 1966. 67. Basu, S. and Kaufman, B., Fed. Proc, 24, 479 (1965). 68. Kaufman, B., Basu, S. and Roseman, S., Methods Enzymol., 8, 365 (1966). 69. Basu, M., Chien,J.L.and Basu, S., Biochem. Biophys. Res. Commun., 60, 1097 (1974). 70. Kijimoto, S., Ishibashi, T. and Makita, Α., Biochem. Biophys. Res. Commun., 56, 177 (1974). 71. Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 240, PC4115 (1965). 72. Steigerwald, J.C., Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 250, 6727 (1975). 73. Kaufman, B., Basu, S. and Roseman, S., Proc. of the Third Int. Symposium on the Cerebral Sphingolipidoses, ed. by S.M. Aronson and B.W. Volk, Pergamo 74. Kaufman, B., Basu (1968). 75. Roseman, S., Chem. Phys. Lipids, 5, 270 (1970). 76. Yip, M.C.M. and Dain, J.A., Biochem. 3.,118,247(1970). 77. Yip, M.C.M. and Dain, J.A., Biochim. Biophys. Acta, 206, 252 (1970). 78. Hildebrand,J.,Stoffyn, P. and Hauser, G.,J.Neurochem.,17,403 (1970). 79. Arce, Α., Maccioni,H.J.and Caputto, R., Fed. Proc, 29, 925 (1970). 80. Moskal, J.R., Gardner, D.A. and Basu, S., Biochem. Biophys. Res. Commun.,61,751 (1974). 81. Kempf, S.F. and Stoolmiller, A.C.,J.Biol. Chem., 251, 7626 (1976). 82. Chien, J.L., Williams, T. and Basu, S., 3. Biol. Chem., 248, 1778 (1973). 83. Ishibashi, T., Kijimoto, S., and Makita, Α., Biochim. Biophys. Acta, 337, 92 (1974). 84. Yeung, K.K., Moskal, J.R., Chien, J.L., Gardner, D.A. and Basu, S., Biochem. Biophys. Res. Commun., 59, 252 (1974). 85. Dawson, G. and Sweeley,CC.,J.Biol. Chem., 247, 410 (1972). 86. Basu, M. and Basu, S.,J.Biol. Chem., 247, 1489 (1972). 87. Basu, M. and Basu, S.,J.Biol. Chem., 248, 1700 (1973). 88. Basu, S., Basu, M. and Chien,J.L.,J.Biol. Chem., 250, 2956 (1975). 89. Basu, S., Basu, M., Moskal, J.R., Chien,J.L.and Gardner, D.A., in Glycolipid Methodology, ed. L.A. Whittig, 1976, p. 123-139. 90. Presper, K.A., Basu, M. and Basu, S., Fed. Proc, 35, 1441 (1976). 91. Pacuszka, T. and Koscielak,J.,Eur.J.Biochem., 64, 499 (1976). 92. Presper, K.A., Basu, M. and Basu, S., Proc. Natl. Acad. Sci., U.S.A., 75, 789 (1978). 93. Watanabe, K., Laine, R.A. and Hakomori, S., Biochemistry, 14, 2725 (1975). 94. Slomiany, B.L. and Slomiany, Α., Biochim. Biophys. Acta, 486, 531 (1977). 95. Gardas, A. and Koscielak,J.,FEBS Lett., 42, 101 (1974).
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March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4 Endo-β-acetylglucosaminidases — Their Metabolic Role in Disease Processes and Their Use in the Study of Glycoprotein Structure and Function FRANK MALEY, ANTHONY L. TARENTINO, and ROBERT B. TRIMBLE New York State Department of Health, Division of Laboratories and Research, Albany, NY 12201
In the course of studie tides from RNase Β an tinase extracts (1, 2) capable of hydrolyzing the di-N-acetyl chitobiosyl linkage of oligosaccharides to yield the following products: R-GlcNAc + GlcNAc(Man)(GlcNAc) where R = Asn; Asnpeptide, Asn-protein, and x = 5 or greater, while y = 0 to 4. This enzyme was called endo-ß-N-acetylglucosaminidase-H (endo-H) because its specificity appeared to be directed towards substrates composed of at least five mannose residues. Further investigation of these initial crude preparations from Streptomyces plicatus revealed them to contain two enzymes, one with a specificity for long chain mannosyl oligosaccharides, and the other with a primary specificity for short chain oligosac charides such as (Man)1(GlcNAc)Asn (2). The latter enzyme, which can be separated from endo-H by gel filtration (Figure 1), has been designated endo-L and will be discussed later. We realize now that it was the presence of this enzyme in our early endo-H preparations that enabled us to affect the isolation of Manβ1-4GlcNAc and to clearly demonstrate for the first time that the core sequence of most Asn-associated glycopeptides is composed of this disaccharide (1, 3, 4). With the former enzyme, designated endo-H, it was shown (1, 5) that the ovalbumin oligosaccharide chain is linked to the distal GlcNAc of the di-N-acetyl chitobiosyl unit and not to the primary GlcNAc as originally believed (6). These high mannosyl or simple oligosaccharides, such as those associated with ovalbumin, ribonuclease B, deoxyribonuclease, carboxypeptidase Y, mungbean nuclease, invertase, immunoglobulin M, and thyroglobulin, were shown by us (6, 7) to be substrates for endo-H and stand in contrast to the resistant "complex" oligosaccharides associated with most immunoglobulins (8, 9, 10) and viral glyco proteins (11) which are composed of a core sequence of Manal "Man 31+4G1 cNAc 31+4G1 cNAc-Asn Manal x
y
2
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where fucose i s u s u a l l y l i n k e d to the f i r s t GlcNAc. The manner i n which t h i s u n i t i s synthesized remains to be determined, but i t appears to r e s u l t from the p r o c e s s i n g of l a r g e r molecular weight o l i g o s a c c h a r i d e chains that are r i c h i n mannose (12, 13). I t has become apparent that enzymes such as endo-H and a r e l a t e d endoglycosidase from Diplococcus pneumoniae, endo-D (14), w i l l provide v a l u a b l e a s s i s t a n c e i n a s s e s s i n g both the s t r u c t u r e and f u n c t i o n of g l y c o p r o t e i n s , t h e i r mechanism of b i o s y n t h e s i s and t h e i r p o t e n t i a l r o l e i n disease processes. But, because of the i n c r e a s i n g i n d i s c r i m i n a t e use of these enzymes without regard to t h e i r s p e c i f i c i t y of a c t i o n and t h e i r attendant contaminants, erroneous c o n c l u s i o n s may r e s u l t . I t i s f o r t h i s reason that I would f i r s t l i k e to review some of the p r o p e r t i e s of these enzymes which are o f t e n overlooked, and to i n c l u d e some of our recent f i n d i n g s which would appear to extend the range of a c t i v i t y of endo-H. As shown i n F i g u r e H r e l a t i v e to endo-D, i t i s c l e a r that endo-H hydrolyzes longer mannosyl o l i g o s a c c h a r i d e s than endo-D, an enzyme which has a much greater s p e c i f i c i t y f o r the branched complex o l i g o s a c c h a r i d e core (Man)3(GlcNAc)2Asn (15). In c o n t r a s t to endo-D, endo-H can e f f e c t i v e l y hydrolyze o l i g o s a c c h a r i d e chains w i t h as many as 50 or more mannosyl r e s i d u e s (j6) and even the complex core r e g i o n , but at a r a t e that i s s e v e r a l orders of magnitude lower than t h a t a f f e c t e d by endo-D, p r o v i d i n g fucose i s not present on the proximal N-acetylglucosamine of the core r e g i o n (Figure 3 ) . As i n d i c a t e d , the presence of fucose completely impairs endo-H s a l r e a d y low a c t i v i t y against the complex core o l i g o s a c c h a r i d e , but has no e f f e c t on endo-D a c t i v i t y . Because of endo-H s c a p a c i t y to r e l i e v e s p e c i f i c g l y c o p r o t e i n s of t h e i r o l i g o s a c c h a r i d e u n i t s , i t can be a v a l u a b l e asset i n e s t i m a t i n g molecular weights of g l y c o p r o t e i n s by SDS-acrylamide g e l e l e c t r o p h o r e s i s , a technique that o f t e n y i e l d s i n a c c u r a t e results with glycoproteins. Thus, by c a r e f u l l y u t i l i z i n g endo-H i n the removal of o l i g o s a c c h a r i d e s from i n v e r t a s e (16), a p r o t e i n w i t h a mass that i s 50% carbohydrate, i t could be shown that t h i s enzyme c o n s i s t s of two i d e n t i c a l s u b u n i t s , each w i t h a molecular weight of 61,000 (Figure 4) a f t e r s u b t r a c t i o n of the remaining N-acetylglucosamines. Incomplete removal of the carbohydrate, however, g i v e s the appearance of two nonequivalent c h a i n s , one w i t h a molecular weight of 65,000 and the other of 68,000 (Figure 5). But s i n c e endo-H i s s t a b l e i n 0.5% SDS, the o l i g o s a c c h a r i d e chains of the i n v e r t a s e molecule can be made completely a c c e s s i b l e to endo-H and almost a l l of i t s 18 o l i g o s a c c h a r i d e chains removed (Table I ) . In the case of CPase Y , four o l i g o s a c c h a r i d e chains were found to be a s s o c i a t e d w i t h a 51,000 d a l t o n monomer (Table I I ) . ?
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40
60
100
80 FRACTION NUMBER
Figure 1. Separation of S. plicatus endo-L (O) and endo-H (±) on a Sephadex G-100 column (1.5 X 220 cm). For further details see Ref. 2. (ao)„-Asn (GlcNAd2(Man)3
100 Asn ( G l c N A c ) 2 (Man) 5
Asn ( G l c N A c ^ (Mon) 6
Asn ( G l c N A c ) 4 (Man) 6
TMan) 5 GlcNAc-GlcNAc-ol
(aa) x -Asn(GlcNAc) 2 (Man) 3
3
0
I
HOURS Biochemical and Biophysical Research
Communications
Figure 2. A comparison of the rates of hydrolysis of various oligosaccharides by endo-H and endo-D. The described substrates were incubated for the indicated times with (A) 2.5 ng of pure endo-H or (B) 0.5 fig of protein containing endo-D. Adapted from Tarentino and Maley (15).
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Endo-β-acetyl^ucosaminidases
(αα) χ -Asn(6lcNAc)2 ( Μ α η ) 3
Biochemical and Biophysical Research Communications
Figure 3. The rate of hydrolysis of ( Fuc)t( Man ) ( GlcNAc ) Asn-(aa )x and its defucosylated derivative by endo-H. The' [ C]-N-acetylated derivatives were incubated with 10 times more endo-H than those in Figure 1. Adapted from Tarentino and Maley (15). 3
2
u
HOURS
Journal of Biological Chemistry
Figure 4. Gel electrophoresis of native (lanes 1-5) and S-carboxymethyhted (SCM) invertase (lanes 7—11) after incubation with endo-H. Endo-H (0.5 μ-g, lane 6) was used to treat 2 mg of native or S-carboxymethylated invertase in a 1 mL reaction mixture. Aliquots were electrophoresed in a flatplate discontinuous SDS-pohjacrijlamide el system. Adapted from Trimble and ialey (16,), which should be referred to for further details.
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TABLE I Carbohydrate content of i n v e r t a s e
Preparation
Native
invertase
(CM)-invertase
Enzyme treatment
preparations
GlcN
Chains/ holoenzyme
Man
mol/120,000 g p r o t e i n 18 35.8 570 4 89 22.3 21.9 18 4
None Endoglycosidase Endoglycosidase + a-mannosidase None Endoglycosidas
35.9 18.9
1 cm A l l values are based on an A:280 of molecular weight of 120,000.
565 16
18 1
2.25 and a holoenzyme
Journal of Biological Chemistry
TABLE I I Carbohydrate content of carboxypeptidase Y f r a c t i o n s before and a f t e r endoglycosidase treatment
Enzyme p r e p a r a t i o n
Native Endoglycosidase-treated Denatured and endoglycosidase-treated
CPase Y molar r a t i o Mannosyl GlcN Man chains 7.90 4.95 3.72
55 15 0
4 1 0
Based on a peptide molecular weight of 51,000.
Biochemical and Biophysical Research
Communications
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Endo-β-acetylglucosaminidases
91
A l t e r n a t i v e l y , molecular weights of the r e l e a s e d o l i g o s a c charides can be estimated by B i o g e l P-4 chromatography p r o v i d i n g appropriate standards are used ( Γ 7 ) . The s i g n i f i c a n c e of the r o l e of carbohydrate attached to p r o t e i n has been of great concern for some time and while removal of the carbohydrate has not g r e a t l y i n f l u e n c e d the enzymic and p h y s i c a l p r o p e r t i e s of most of the g l y c o p r o t e i n s we and others have s t u d i e d , an a l t e r a t i o n i n such p h y s i c a l parameters as s o l u b i l i t y has been noted, p a r t i c u l a r l y i n the case of i n v e r t a s e which possesses such a high mass of t h i s m a t e r i a l . Recently i t has been shown by Wang and H i r s (18) that carbohydrate i n f l u e n c e s the spec t r a l p r o p e r t i e s of ovine RNase B. Even more r e c e n t l y , we have found the s t a b i l i t y of carbohydrate depleted i n v e r t a s e to be markedly a l t e r e d at pH 4.0 r e l a t i v e to n a t i v e i n v e r t a s e (Figure 6). This parameter i s a l s o r e f l e c t e d i n the decreased c a p a c i t y of the carbohydrate depleted enzyme to renature when denatured by g u a n i dine h y d r o c h l o r i d e , suggestin i n f l u e n c e s the c a p a c i t y Because of the r a t h e r high content of carbohydrate i n t h i s enzyme, i t i s d i f f i c u l t to extrapolate these s t u d i e s to other g l y c o p r o t e i n s with considerably lower amounts of carbohydrate. Caution must be e x e r c i s e d i n i n t e r p r e t i n g these f i n d i n g s however, and the p o t e n t i a l for contaminants a s s o c i a t e d with even h i g h l y p u r i f i e d endoglycosidases makes t h i s even r i s k i e r . Thus, even the presence of small q u a n t i t i e s of exoglycosidases or p r o teases can g r e a t l y i n f l u e n c e the r e s u l t s obtained c o n s i d e r i n g the lengthy incubations often used by i n v e s t i g a t o r s . Witness the case of endo-D which was i n i t i a l l y b e l i e v e d to r e l e a s e complex o l i g o saccharides (19) u n t i l i t was r e a l i z e d on further p u r i f i c a t i o n that p e r i p h e r a l sugars as s i a l i c a c i d , g a l a c t o s e , and N - a c e t y l g l u cosamine must f i r s t be r e l e a s e d by contaminating exoglycosidases before the (Man^GlcNAc c h a i n could be r e l e a s e d (14). Even when p u r i f i e d about 4,000 f o l d to apparent homogeneity by the procedure shown i n Table I I I , endo-H contains t r a c e s of p r o t e o l y t i c a c t i v i t y which are not r e a d i l y apparent unless more s e n s i t i v e techniques than o r d i n a r i l y used are a p p l i e d , such as measuring the r e l e a s e of r a d i o a c t i v i t y from hemoglobin l a b e l e d with l ^ C - l a b e l e d g l y c i n e e t h y l e s t e r . Thus, i t i s shown i n F i g u r e 7, which presents a G-75 e l u t i o n p r o f i l e , the l a s t step i n the p u r i f i c a t i o n of endo-H, that small t r a c e s of p r o t e o l y t i c a c t i v i t y are s t i l l present i n the enzyme peak. Denatured CPase Y i s par t i c u l a r l y s e n s i t i v e to t h i s p r o t e o l y t i c a c t i v i t y and i t i s shown i n F i g u r e 8 that t h i s p r o t e i n i s hydrolyzed to v a r y i n g degrees by d i f f e r e n t f r a c t i o n s from the endo-H peak, even though the enzyme appears to be homogenous at t h i s stage. However, treatment of those f r a c t i o n s c o n t a i n i n g p r o t e o l y t i c a c t i v i t y with phenylmethane s u l f o n y l f l u o r i d e almost completely e l i m i n a t e s t h i s contaminant. One must be cautious i n a s s e s s i n g the molecular weight of l a b e l e d o l i g o s a c c h a r i d e s , that glycopeptides are not i n r e a l i t y being considered.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 5. Electrophoresis of native invertase treated with endo-H. The systems used for digestion and electrophoresis were simifor to those in Figure 4. The two forms of the enzyme represent incompletely removed oligosaccharide chains associated with the subunits.
HOURS Figure 6. Stability of native and carbohydrate (CHÔ)-depleted invertase maintained at pH = 4.0. Each enzyme form (1 fig) was incubated in 1 mh of 0.01M sodium citrate pH = 4.0 at 4°C for the indicated times and then assayed (F. K. Chu and F. Maley, in preparation).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
III
50
37
19.7
67
5.5
0.13
72
100
<%)
Recovery
T h i s procedure represents a m o d i f i c a t i o n from that p r e v i o u s l y d e s c r i b e d (2), and w i l l appear i n a future volume of Methods i n Enzymology.
a S t e p 1 m a t e r i a l was prepared i n 1 0 - l i t e r batches. Steps 2 through 4 were performed on the zinc paste from 30 l i t e r s of c u l t u r a l f i l t r a t e . Step 5 was performed by combining two preparations from the preceeding s t e p .
69
Sephadex G-75, pH 8.45
5.
3.5
94
17
404
SP-Sephadex C-25, pH 4.6
4. 29
126
996
680
DE-52 C e l l u l o s e , pH 8.45
3.
0.02
136
6,750
1,300
Ammonium s u l f a t e , 0.4-0.9
189
34,430
60,300
2.
0.0053
Activity (units)
Protein (mg)
Specific activity (units/mg)
plicatus
Volume (ml)
C u l t u r a l f i l t r a t e and zinc p r e c i p i t a t i o n
step
1.
Purification
P u r i f i c a t i o n of endo-3-N-acetylglucosaminidase H from Streptomyces
TABLE
94
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES 1.51
Figure 7. Assessment of residual proteo lytic activity present in the Sephadex G-75 elution profile from Step 5 of Table III. The substrate used to measure proteolysis was [ C]-glycine ethylester hemoglobin (13,000 cpm/mg protein). The peak of enzyme activity was associated with a single band of protein on polyacrylamide gel electroprhoresis. ( Ο — θ ) Endo-H ac tivity (cpm χ 10 ) or (A—^protease ac tivity (cpm X JO ). 14
4
2
20
40
60
80
FRACTION NUMBER (3 ml)
Figure 8. Assay for proteolytic activity in various fractions from the elution profile of Figure 7. Carboxypeptiaase Y (1 mf>) was incubated with 0.2 unit of endo-H from the indicated fractions in a 1 mh of reaction mixture for 4 hr at 37°C. Equal aliquots from these reactions were denatured with sodium dodecul sulfate and electrophoresed on SDS-polyacrylamide Eel as described in Trimble and Maley (7). The migration distances of untreated (-\-CHO) ana depleted (-CHO) carboxypeptiaase Y and endo-H are indicated in the first two lanes at the left.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
Endo-β-acetylglucosaminidases
95
From what was i n d i c a t e d i n Figure 2, endo-H appears most a c t i v e with substrates such as (Man)5(GlcNAc)2Asn, but because of the u n a v a i l a b i l i t y u n t i l now of compounds such as (Man)4(GlcNAc)2~ Asn and (Man)2(GlcNAc)2Asn, i t was not p o s s i b l e to determine the lower l i m i t s of the enzyme's s p e c i f i c i t y . R e c e n t l y , however, T a i et a l . (20) i s o l a t e d (Man)4(GlcNAc)2Asn from an ovalbumin d i g e s t by use of the Montgomery column (6^) and found that t h i s compound l i k e (Man)5(GlcNAc)2Asn was an e x c e l l e n t substrate for endo-H. By t r e a t i n g (Man)5(GlcNAc)2Asn with α - m a n n o s i d a s e for l i m i t e d time p e r i o d s , we have been able to i s o l a t e (Man)4(GlcNAc)2Asn and (Man)β(GlcNAc)2Asn through the use of B i o g e l P-4 chromatography. It was i n i t i a l l y b e l i e v e d , from the B i o g e l P-4 e l u t i o n p r o f i l e , that the l a t t e r compound was (Man)2(GlcNAc)2Asn, but by comparing i t s e l u t i o n time with other g l y c o s y l - A s n d e r i v a t i v e s on the amino a c i d analyzer (Figure 9 ) , i t was e s t a b l i s h e d that t h i s o l i g o s a c charide was composed of (Man)3(GlcNAc)2Asn (Figure 9A, b ) . The i n s e r t to Figure 9A e s t a b l i s h e graphic procedure for c h a r a c t e r i z i n up to s i x mannosyl r e s i d u e s . M e t h y l a t i o n of (Man)4(GlcNAc)2Asn revealed t h i s compound to d i f f e r from that i s o l a t e d by T a i et a l . (19) i n that the p e r i p h e r a l mannosyl linkage was al+6 (Figure 10 and Table IV) instead of al->3 as shown by these i n v e s t i g a t o r s . In contrast to the branched s t r u c t u r e of (Man)3(GlcNAc)2Asn, a s s o c i a t e d with the core of the complex o l i g o s a c c h a r i d e s , that i s o l a t e d from the α - m a n n o s i d a s e d i g e s t i o n of (Man)5(GlcNAc)2Asn, was determined by methylation (Table IV) and periodate o x i d a t i o n to be l i n e a r and to possess the p e r i p h e r a l s t r u c t u r e shown i n F i g u r e 10. Both (Man)4(GlcNAc)2Asn and l i n e a r (Man)3(GlcNAc)2Asn appear to be hydrolyzed as r a p i d l y by endo-H as i t s most a c t i v e s u b s t r a t e , which to date i s (Man)5(GlcNAc)2Asn, and c o n t r a s t s sharply w i t h the rather slow h y d r o l y s i s by t h i s enzyme of branched (Man)3(GlcNAc)2Asn (Figures 2 and 3). By u t i l i z i n g a modified Smith degradation on (Man)5(GlcNAc)2Asn (21, 22), (Man)2(GlcNAc)2Asn could be i s o l a t e d and v e r i f i e d by compositional a n a l y s i s , methyla t i o n (Table I V ) , and chromatography (Figure 9B). T h i s compound was hydrolyzed at only h a l f the r a t e of branched (Man)3(GlcNAc)2~ Asn, while (Man)1(GlcNAc)2Asn i s probably not hydrolyzed at a l l by endo-H. Thus, the statement i n our a b s t r a c t that (Man)2(GlcNAc)2Asn i s as good a substrate as (Man)5(GlcNAc)2Asn i s i n e r r o r and should be corrected by r e p l a c i n g the former compound with l i n e a r (Man)3(GlcNAc)2Asn. (Man)1(GlcNAc)2Asn, however, could be very e f f e c t i v e l y hydrolyzed to Man31->4GlcNAc + GlcNAc-Asn by endo-L, the enzyme r e f e r r e d to e a r l i e r as enabling us to i s o l a t e and to c h a r a c t e r i z e t h i s d i s a c c h a r i d e (1). That t h i s enzyme i n not c h i t o b i a s e i s i n d i c a t e d by i t s i n a b i l i t y to hydrolyze (GlcNAc) 2 Asn. Endo-L has now been p u r i f i e d about 300 f o l d and possesses proper t i e s d i s t i n c t l y d i f f e r e n t from endo-H, not the l e a s t of which i s t h e i r substrate s p e c i f i c i t i e s . Other d i f f e r e n c e s are a s s o c i a t e d with the higher molecular weight of endo-L and i t s sharper pH optimum (23). A comparison of these enzymes and t h e i r capacity to
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
96
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
175
35
52.5
70
0.1 M Ν α - C I T R A T E (pH 2.60), ml Figure 9.
Chromatographic separation of glycosyl asparagine derivatives on the amino acid analyzer using AG-50 X-4 resin.
The various stands used (prepared from ovalbumin) are indicated by the dashed line and repre sent (Man) (GlcNAc) Asn, 1-2-6; (Man) (GlcNAc) Asn, 1-2-5; (Man) (GlcNAc) Asn 1-2-1; (GlcNAc) Asn, 1-2; GlcN Ac-Asn, 1-1. Peaks a and b resulted from a limited a-mannosidase digestion of 1-2-5 as shown in Figure 10; peak c in Β was prepared by the periodate oxidation of 1-2-5; peak d in C was obtained by treating 1-2-2 with periodate. The elution times (t), measured at peaks of various glycosyl-Asn derivatives, were plotted as shown in the insert vs. the number of mannose residues in the compounds. As indicated, compounds a, b , and c fall on the line at 4, 3, and 2 mannose residues respectively, which is consistent with their mannosyl content (Table IV). The proposed mannosyl linkages shown in Figure 10 were obtained from the methylation data in Table IV. More specific details on the methodology used are in prepa ration. 6
2
5
2
t
2
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 1.00
(Man) 2 (GlcNAc) 2 A s n b
endo-H
1.0
0.84
2.2
1.4
1.1
1.3 1.0
1.0
Methylated mannitol products (2,3,4,6) (2,3,4) (2,4,6) (2,4)
s u b s t r a t e s for
'Periodate o x i d a t i o n product from (Man)r (GlcNAc)οAsn.
Prepared from IgM (9) ; R = p e p t i d e .
1.96
3.00
2.00
1.55
(Man) 3 (GlcNAc) 2 - R a 1.80
2.84
1.82
1.00
4.06
1.85
(Man) 3 (GlcNAc) 2 A s n
Man
Analysis GlcNAc
1.00
Asn
of compounds tested as
(Man) 4 (GlcNAc) 2 A s n
Isolated compound
Composition
TABLE IV
98
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
hydrolyze s p e c i f i c dansylated substrates prepared from ovalbumin (Figure 11) r e v e a l s that endo-L i s not very e f f e c t i v e against sub s t r a t e s c o n t a i n i n g more than two r e s i d u e s of mannose. In a s i m i l a r v e i n , endo-H i s not e f f e c t i v e against compounds c o n t a i n i n g two mannosyl residues or l e s s . We cannot be c e r t a i n at present whether the small amount of a c t i v i t y seen with (Man)3(GlcNAc)2Asn as s u b s t r a t e i s n a t i v e to endo-L and i s not due to traces of endo-H. There i s , however, a greater degree of confidence that homogenous endo-H decreases i n a c t i v i t y by 10,000 f o l d i n going from l i n e a r (Man)3(GlcNAc)2Asn to (Man)2(GlcNAc)2Asn. These recent s p e c i f i c i t y s t u d i e s suggest the need for c a u t i o n i n e s t i m a t i n g o l i g o s a c c h a r i d e s i z e based on the s e n s i t i v i t y or r e s i s t a n c e of glycopeptides to endo-H. Following our c h a r a c t e r i z a t i o n of the p r o p e r t i e s of endo-H (2, 6, 15) and that of endo-D by Koide and Muramatsu (14) a number of other u s e f u l endoglycosidases have been i d e n t i f i e d . Thus, an endo-3-N-acetyl-galactosaminidas f i l t r a t e s of Diplococcu from E s c h e r i c h i a f r e u n d i i (25), two endo-3-N-acetylglucosaminidases from C l o s t r i d i u m perfringens (26), one with s l i g h t l y d i f f e r ent s p e c i f i c i t y than endo-H, and more r e c e n t l y an amidase from almond emulsin (27). The amidase, although not p u r i f i e d to a s i g n i f i c a n t extent, possesses the i n t r i g u i n g property of r e l e a s i n g o l i g o s a c c h a r i d e s from a stem bromelein glycopeptide by h y d r o l y z i n g the glucosaminyl asparagine bond. If t h i s enzyme i s more gener a l l y a p p l i c a b l e , i t should be extremely u s e f u l i n the study of glycopeptides and p o s s i b l y g l y o c p r o t e i n s , but not enough i n known about i t s s p e c i f i c i t y at the present time. As far as the existence of an animal endoglycosidase with p r o p e r t i e s s i m i l a r to those of endo-H or endo-D, i t s presence can be i n f e r r e d from the s t r u c t u r e of o l i g o s a c c h a r i d e s excreted i n the u r i n e and present i n the t i s s u e s of animals with c e r t a i n lysosomal storage d i s e a s e s . Thus, mannosyl o l i g o s a c c h a r i d e s with a s i n g l e N-acetylglucosamine on the reducing end were found i n the u r i n e and t i s s u e of b l a c k angus c a t t l e (28) and humans (29) a f f l i c t e d with α - m a n n o s i d o s i s . O l i g o s a c c h a r i d e s c o n s i s t i n g of 7 to 10 sugar r e s i d u e s with the complex t e t r a s a c c h a r i d e core of (Man^GlcNAc have a l s o been found i n p a t i e n t s with GM]^ or GM2 g a n g l i o s i d o s i s (30, 31, 32), and f i b r o b l a s t s from p a t i e n t s with α - f u c o s i d o s i s (32) apparently accumulate the d i s a c c h a r i d e Fucal-*6GlcNAc, a d i s a c c h a r i d e found at the reducing end of many "complex" type oligosaccharides. Whether these products accumulate due to the above s p e c i f i c exoglycosidase d e f i c i e n c i e s combined with cleavages a f f e c t e d by a s i n g l e endoglycosidase i s not known, but we have described an enzyme i n animal t i s s u e s p a r t i a l l y capable of e x p l a i n i n g these f i n d i n g s (33). The enzyme was p u r i f i e d over 1,500 f o l d from hen-oviduct and as shown i n F i g u r e 12 hydrolyzed [ l ^ C ] - N - a c e t y l a t e d g l y c o s y l - A s n d e r i v a t i v e s derived from ovalbumin i n a manner s i m i l a r to that described for endo-H. As an added bonus, t h i s enzyme p r e p a r a t i o n
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
MALEY ET AL.
Endo-β-acetylglucosaminidases
Manal - ^ 6 Μ β η β 1 ^ » 4 G 1 c N A c 0 1
•>
4G1 c N A c - A s n
10.
b) H
+
Manal , ^ 6 Manal
^
Manal ^
ManÊl Manal
Manal
4G1 cNAcS1
4G1 c N A c - A s n
^
r
6Manal
a-Mannosidase
Man 31 <
M a n a l ^ » 3Manal
-•6ManBl.
Manal
Figure 10. Proposed structures of compounds prepared from (Man) (GlcNAc) Asn by periodate oxidation and limited α-mannosidase digestion. The structures proposed are based on compositional analyses, in addition to the methylation and periodate oxidation data presented in Figure 9 and Table IV. R = (GlcN Ac) -Asn. 5
2
2
CLEAVAGE OF OV-OLIGOSACCHARIDES BY E N D O - L AND -H Asn(GlcNAc) Man 2
AsniGlcNAcfeMai^
AsnfêlcNAcfeMons Asn(GlcNAc)4Mûn
6
OR ENDO-L
~
"
ENDO-H
-
+
+ -
-
- + +
-
-
- +
-
-
+
-
+
-
+
-
-
Figure 11. Studies on the substrate specificity of endo-H and endo-L. The corresponding dansyl derivatives of the compounds indicated were treated with endo-L and endo-H as shown, and aliquots were chromatographed ascendingly on Whatman 3-MM with butanol-ethanol-water (2:1:1).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Journal of Biological Chemistry
Figure 12. Substrate specificity of hen oviduct endoglycosidase. The indicated [ C]-N-acetylated derivatives were etectrophoresed on paper before and after treatment with this enzyme preparation, followed by the preparation of a radioautogram. Adapted from Trimble et al. (23) which should be consulted for further details. 14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
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a l s o hydrolyzed the complex core glycopeptide c o n t a i n i n g fucose, a property of endo-D but not of endo-H. A l s o c h a r a c t e r i s t i c of endo-D i s the f i n d i n g that the complex branched g l y c o p e p t i d e , (Man)3(GlcNAc)2Asn, i s hydrolyzed at a f a s t e r r a t e than (Man) 6 (GlcNAc)4Asn (33), the reverse of that obtained with endo-H (Figure 2 ) . I f an enzyme s i m i l a r to t h i s one i s present i n animal t i s s u e s from other s p e c i e s , the type of o l i g o s a c c h a r i d e s a s s o c i ated with the above mentioned genetic d i s o r d e r s can e a s i l y be rationalized. Through the use of a new assay we have developed where the mannosyl non-reducing end of g l y c o s y l - A s n d e r i v a t i v e s , such as (Man)5(GlcNAc)2Asn-dansyl, are l i g h t l y o x i d i z e d with p e r i odate and then reduced with sodium b o r o t r i t i d e , endoglycosidase a c t i v i t y has been found i n s e v e r a l r a t t i s s u e s , as w e l l as i n Tetrahymena p y r i f o r m i s (34). An advantage of t h i s s u b s t r a t e i s the r e s i s t a n c e of the l a b e l e d d i o l to α - m a n n o s i d a s e , an enzyme which could y i e l d a f a l s e p o s i t i v e endoglycosidase a c t i v i t y i n crude t i s s u e e x t r a c t s . Although an animal genetic d i s o r d e r i m p l i c a t i n g endo-3-Nacetylglucosaminidase has not been reported y e t , the question a r i s e s as to what might be expected i f an organism i s d e f i c i e n t i n t h i s enzyme. In a l l p r o b a b i l i t y t h i s defect would be d i f f i c u l t i f not impossible to assess s i n c e the numerous types of e x o g l y c o s i dases present i n animal t i s s u e s would l i m i t the accumulation of long chain o l i g o s a c c h a r i d e s a s s o c i a t e d with glycopeptides i n v o l v ing glucosaminyl-Asn l i n k a g e groups. That i s unless α - m a n n o s i d a s e was a l s o d e f i c i e n t , i n which case o l i g o s a c c h a r i d e s with GlcNAc on the reducing end would not be found and glycopeptides with mannosy 1 - r i c h o l i g o s a c c h a r i d e s should accumulate. At present, the r o l e of endo-H and other s i m i l a r l y r e l a t e d enzymes appears to be r e s t r i c t e d to that of degrading g l y c o p r o t e i n s and g l y c o p e p t i d e s , but t h e i r p o t e n t i a l c o n t r i b u t i o n to the b i o s y n t h e s i s and processing of g l y c o p r o e t i n s should not be ignored. In any event, the d i s c o v e r y of the endoglycosidases has added a new dimension to the study of g l y c o p r o t e i n s , which h o p e f u l l y w i l l continue to provide new and important information on t h e i r s t r u c t u r e and f u n c t i o n .
Acknowledgements We would l i k e to express our a p p r e c i a t i o n to D r . Thomas H. Plummer, J r . and Dr. F r e d e r i c k K. Chu for t h e i r a i d with some aspects of t h i s study.
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Literature Cited 1. Tarentino, A. L., Plummer, T. H., Jr., and Maley, F., J. Biol. Chem. (1972) 247, 2629. 2. Tarentino, A. L., and Maley, F., J. Biol. Chem. (1974) 249, 811. 3. Sukeno, T., Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., Biochem. Biophys. Res. Commun. (1971) 45, 219. 4. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., J. Biol. Chem. (1973) 248, 5547. 5. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., J. Biol. Chem. (1974) 249, 818. 6. Huang, C.-C., Mayer, H.-E., Jr., and Montgomery, R., Carbohydrate Res. (1970) 13, 127. 7. Trimble, R. B., and Maley, F., Biochem. Biophys. Res. Commun. (1977) 78, 935. 8. Baeziger, J., and Kornfeld 7260. 9. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., Biochemistry (1975) 14, 5516. 10. Kornfeld, R., and Kornfeld, S., Annu. Rev. Biochem. (1976) 45, 217. 11. Burke, D. J., and Keegstra, K., J. Virol. (1976) 20, 676. 12. Robbins, P. W., Hubbard, S.C.,Turco, S. J., and Wirth, D. F., Cell (1977) 12, 893. 13. Tabas, I., Schlesinger, S., and Kornfeld, S., J. Biol. Chem. (1978) 253, 716. 14. Koide, Ν., and Muramatsu, T., J. Biol. Chem. (1974) 249, 4897. 15. Tarentino, A. L., and Maley, F., Biochem. Biophys. Res. Commun. (1975) 67, 455. 16. Trimble, R. B., and Maley, F., J. Biol. Chem. (1977) 252, 4409. 17. Etchinson, J. R., Robertson, J. S., and Summers, D. F., Virology (1977) 78, 375. 18. Wang, F.-F.C.,Hirs, C. H. W., J. Biol. Chem. (1977) 252, 8358. 19. Muramatsu, T., Atkinson, P. H., Nathenson, S. G., and Ciccarini, C., J. Mol. Biol. (1973) 80, 781. 20. Tai, T., Yamashita, Κ., and Kobata, Α., Biochem. Biophys. Res. Commun. (1977)78,434. 21. Montgomery, R., Wu, Y.-C, and Lee, Y. C., Biochemistry (1965) 4, 578. 22. Garner, E. F., Goldstein, I. J., Montgomery, R., and Smith, F., J. Am. Chem. Soc. (1958) 80, 207. 23. Trimble, R. B., Tarentino, A. L., and Maley, F., in preparation. 24. Umenoto, J. Bhavanandan, V. P., and Davidson, Ε. Α., J. Biol. Chem. (1977) 252, 8609. 25. Fukuda, Μ. Ν., and Matsumura, G., J. Biol. Chem. (1976) 251, 6218.
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26. Tai, T., Yamashita, Κ., Ito, S., and Kobata, Α., J. Biol. Chem. (1977) 252, 6687. 27. Takashi, N., Biochem. Biophys. Res. Commun. (1977) 76, 1194. 28. Lundblad, Α., Nilsson, B., Nordén, Ν. Ε., Svensson, S., Ôckerman, P.-Α., and Jolly, R. D., Eur. J. Biochem. (1975) 59, 601. 29. Nordén, Ν., Lundblad, Α., Svensson, S., and Autio, S., Biochemistry (1974) 15, 871. 30. Wolfe, L. S., Senior, R. G., and Kin, Ν., J. Biol. Chem. (1974) 249, 1828. 31. Kin. Ν., and Wolfe, L. S., Biochem. Biophys. Res. Commun. (1974) 59, 837. 32. Tsay, G.C.,and Dawson, G., Biochem. Biophys. Res. Commun. (1975) 63, 807. 33. Tarentino, A. L. and Maley F. J Biol Chem (1976) 251 6537. 34. Tarentino, A. L., Maley, , (1977) , 185. RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5 Mucous Glycoproteins in Cystic Fibrosis THOMAS F. BOAT and PIWAN CHENG Department of Pediatrics, Rainbow Babies and Childrens Hospital, Case Western Reserve University, School of Medicine, Cleveland, OH 44106 Cystic fibrosis (CF) Caucasians, causes muc progressiv g children and young adults. It also is responsible for intestinal obstruction at birth (meconium ileus), pancreatic insufficiency with maldigestion and failure to grow, and obstructive liver disease in this age group. Furthermore, obliteration of the vas deferens causes aspermia and infertility in most males with CF. Individuals with CF secrete sweat which contains excessive amounts of chloride and sodium. Diagnostic criteria include detection of elevated chloride levels in sweat of individuals with typical pul monary, gastrointestinal, or genital manifestations of the disease (1). The pathophysiology of CF has been the subject of extensive investigation (2). However, the basic defect responsible for the disease has not been identified. Claims have been made by several investigators that circulating factors which can be detected by inhibition of oyster gill (3) or rabbit tracheal (4) ciliary motil ity, or secreted factors which inhibit sodium reabsorption by ductal epithelium in sweat and salivary glands (5), may be related closely to the gene defect. Alteration of short circuit current across rat intestinal epithelium, inhibition of sugar and amino acid transport by intestinal epithelium, augmentation of leukocyte degranulation, inhibition of phagocytosis by alveolar macrophages, and inhibition of debranching enzyme activity have also been as cribed to circulating or secreted "factors" associated with CF. Polyamines, their condensation products (6) and small polycationic peptides (7) have been implicated as "factors" but a substance has not been isolated and characterized which is clearly responsible for any of these effects. Cell membrane properties with the ex ception of increased resistance of fibroblasts to oubain toxic ity (8) appear unaltered. Recent evidence suggests a deficiency of proteolytic activity in CF serum (9) and abnormal interaction of circulatingα -macroglobulinwithproteolyticenzymes(10). Pursuit of initial observations in many of these studies has led to contradictory results, making most studies difficult to inter2
0-8412-0452-7/78/47-080-108$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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p r ê t or f i t i n t o an o v e r a l l p a t h o p h y s i o l o g i c scheme. One poss i b l e e x p l a n a t i o n f o r i n c o n s i s t e n c i e s i n s t u d i e s c a r r i e d out to date i s that CF may be due to genetic abberations at more than one l o c u s . On the other hand the pathogenesis of most c l i n i c a l manif e s t a t i o n s i n CF i s r e l a t i v e l y w e l l understood and appears to be r e l a t e d to two anomalies: 1) abnormal behavior of the mucuss e c r e t i o n s , g i v i n g r i s e to o b s t r u c t i o n , and i n some cases i n f e c t i o n w i t h i n mucus-secreting organs ( l u n g , i n t e s t i n e , pancreas, b i l i a r y t r a c t , s a l i v a r y g l a n d s , and g e n i t o u r i n a r y t r a c t ) , and 2) abnormality of the e l e c t r o l y t e composition of e c c r i n e sweat glands, o c c a s i o n a l l y r e s u l t i n g i n heat p r o s t r a t i o n due to massive s a l t l o s s (2). T h i s review w i l l examine the abnormality of mucous s e c r e t i o n s i n d e t a i l and present current t h i n k i n g about i t s biochemical b a s i s . Emphasis w i l l be given to lung mucus because a i r f l o w o b s t r u c t i o n and lung i n f e c t i o n are r e s p o n s i b l e for most of the m o r b i d i t CF. Mucus from i n d i v i d u a l s with CF g e n e r a l l y i s d e s c r i b e d as abundant and e x c e s s i v e l y s t i c k y . R h é o l o g i e s t u d i e s of lung mucus have been c a r r i e d out l a r g e l y on sputum (11) w i t h a high content of DNA (12) which masks the v i s c o e l a s t i c p r o p e r t i e s of the primary s e c r e t i o n s . While c a r e f u l r h é o l o g i e s t u d i e s of u n i n f e c t ed mucous s e c r e t i o n s with a low DNA content from CF subjects have not been r e p o r t e d , i t i s reasonable to assume that the o b s t r u c t i v e events l e a d i n g to the c l i n i c a l syndrome are the r e s u l t of abnormal p r o p e r t i e s of mucus, and perhaps of the mucous g l y c o p r o t e i n s themselves which seem to be the primary determinant of uninfected dog t r a c h e a l mucus (13). Three p o t e n t i a l causes f o r these abnormal p r o p e r t i e s can be proposed: 1) h y p e r s e c r e t i o n of normal mucus which overloads clearance mechanisms, r e s u l t i n g i n s t a s i s and o b s t r u c t i o n ; 2) inadequately hydrated mucus or mucus with an a l t e r e d i o n content such that g l y c o p r o t e i n complexes are formed which have abnormal r h é o l o g i e p r o p e r t i e s ; or 3) a l t e r a t i o n of chemical p r o p e r t i e s of mucous g l y c o p r o t e i n s r e s u l t i n g i n f o r mation of mucous g e l s with abnormal r h é o l o g i e c h a r a c t e r i s t i c s . Mucus H y p e r s e c r e t i o n : Morphologic examination of s e v e r a l organs from i n d i v i d u a l s with CF shows marked enlargement of the mucuss e c r e t i n g glands (hypertrophy) and a marked increase i n the number of mucus-secreting goblet c e l l s w i t h i n the surface e p i t h e l i u m (hyperplasia) (14). T h i s i s true not only of l u n g , but a l s o of i n t e s t i n e , s a l i v a r y glands, and u t e r i n e c e r v i x . In f a c t , goblet c e l l s appear e a r l y i n the course of the d i s e a s e i n the surface e p i t h e l i u m of v e r y small airways (bronchioles) where they normally do not r e s i d e i n s u b s t a n t i a l numbers. T h i s m e t a p l a s t i c change introduces mucus i n t o airways which are not equipped to handle g e l - l i k e s e c r e t i o n s . Mucus then impacts i n these small airways, producing the e a r l i e s t r e c o g n i z a b l e lung l e s i o n (15). Morphologic evidence f o r a hypersecretory s t a t e i n CF lungs at b i r t h i s c o n t r a d i c t o r y (16,17). However, i t i s c l e a r that duo-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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denal mucus-secreting glands are enlarged and apparently hypera c t i v e at b i r t h (17). T h i s f i n d i n g suggests that increased s e c r e t i o n of mucus may be more than a secondary r e a c t i o n to c h r o n i c i n f e c t i o n or inflammation. A search f o r p a t h o p h y s i o l o g i c determinants of mucous hypers e c r e t i o n u n t i l r e c e n t l y turned up few l e a d s . In the l a s t s e v e r a l y e a r s , s t u d i e s of the e f f e c t of serum on c i l i a r y m o t i l i t y l e d to the observation that l a r g e amounts of "mucus" were r e l e a s ed from c i l i a t e d e p i t h e l i u m i n the presence of CF serum (18,19). Recently Czegledy-Nagy and Sturgess (20) studied t h i s response by scanning e l e c t r o n microscopy and observed that r a b b i t t r a c h e a l e p i t h e l i u m r e l e a s e d more s e c r e t o r y product i n the presence of CF than c o n t r o l serum. These observations are q u a l i t a t i v e and i n a d d i t i o n , products which were assumed to c o n t a i n mucous g l y c o p r o t e i n s were not i d e n t i f i e d . We have developed an assay to assess the e f f e c t of CF serum and other b i o l o g i c a l f l u i d s on the r e l e a s e of mucous g l y c o p r o t e i n G l y c o p r o t e i n s are l a b e l e glucosamine, and t h e i r r e l e a s e i n t o c u l t u r e medium i n the presence of 50% (v/v) CF serum, 50% (v/v) c o n t r o l serum, and no serum i s monitored. P r e l i m i n a r y r e s u l t s show that the presence of serum from any source i n the medium increases the r e l e a s e of l a b e l e d high molecular weight macromolecules which have a carbohydrate composition t y p i c a l of mucous g l y c o p r o t e i n s . Some CF sera t r a n s i e n t l y induce a higher s e c r e t o r y r a t e f o r these l a b e l e d macromolecules than i s achieved i n the presence of age and sexmatched c o n t r o l s e r a , but o v e r a l l we have not yet been able to demonstrate a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t f o r CF sera (Figure 1 ) . At longer exposure periods (1 hour or more) no suggestion of a d i f f e r e n t i a l CF serum e f f e c t on mucous g l y c o p r o t e i n r e l e a s e was found. The nature of the s t i m u l a t o r y e f f e c t i n some CF sera i s not known. Polyamines augment the r e l e a s e of g l y c o p r o t e i n s from canine t r a c h e a l expiants (22) but not human t r a c h e a l e p i t h e l i a l expiants (23). P o l y c a t i o n i c peptides a l s o increase the r e l e a s e of g l y c o p r o t e i n s i n v i t r o from dog t r a c h e a l e p i t h e l i u m (24). P o l y c a t i o n i c substances may i n f l u e n c e mucous g l y c o p r o t e i n synthesis by s t i m u l a t i n g the a c t i v i t y of g l y c o s y l t r a n s f erases at low Mn + ^ concentrations (25) . Whether t h i s occurs with i n t a c t t i s s u e s under p h y s i o l o g i c c o n d i t i o n s i s not known. Expiants of post-mortem t r a c h e o b r o n c h i a l e p i t h e l i u m from CF subjects s e c r e t e mucous g l y c o p r o t e i n s at a higher b a s e l i n e r a t e than do expiants from c h i l d r e n and a d u l t s who have d i e d of nonr e s p i r a t o r y c o n d i t i o n s (26). This observation i s c o n s i s t e n t w i t h an increased mass of mucus-secreting c e l l s i n t i s s u e s of subjects with severe b r o n c h i t i s as i t occurs i n CF, and does not r e f l e c t an increased i n t r i n s i c l e v e l of f u n c t i o n f o r CF s e c r e t i n g u n i t s . Further support f o r t h i s concept comes from autoradiographic s t u d i e s of Neutra (27) which show no d i f f e r e n c e i n the r a t e of movement of l a b e l e d g l y c o p r o t e i n precursors through goblet c e l l s of CF, compared with c o n t r o l , r e c t a l e p i t h e l i a l e x p i a n t s .
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Figure 1. Discharge of SO and Hlabeled glycoproteins by rabbit tracheal expiants in the presence of no serum (C), normal serum (N), and CF serum (CF). 35
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Expiants were cultured in the presence of 2 X 10 dpm of 3 H-6-D-glucosamine and 1 Χ 10 dpm of SO in 2 mh of medium for 24 hr (Fj), rinsed thoroughly with unlabeled me dium and then cultured for 10 min (PJ in 2 mL of unlabeled medium containing no serum or 50% (v/v) serum. Media harvested from P, and P culture periods, including washings, were treated with 5% trichloro acetic acid-1 % phosphotungstic acid and the washed precipitates were solubilized with hydroxide of hyamine-H 0 for scintillation counting (24). Data are expressed as a ratio of the rate of acid-precipitahle label released in P to that in P (56) for each experimental condition. Approximately 80% of labeled macromolecules released by rabbit tracheal explants were high molecular weight sub stances as assessed by BioGel A-5m chroma tography and had a carbohydrate composition typical for mucous glycoproteins (21). Con stant specific activity of discharged glycopro teins was achieved within 24 hr of exposure to medium containing labeled precursors. C vs. Ν; ρ < 0.01. C vs. CF; ρ < 0.01. Ν vs. CF; 0.05
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Sturgess and Reid observed w i t h autoradiographic techniques that the response of ^ s e c r e t o r y index ( p r o p o r t i o n of c e l l s show ing i n c o r p o r a t i o n of Η - g l u c o s e and movement of l a b e l to the lumenal surface) to cholinomimetic s t i m u l a t i o n was higher f o r hypertrophic glands of CF t r a c h e a l expiants than f o r glands from n o n - b r o n c h i t i c tracheas (28) . However, d i r e c t measurement of the s e c r e t o r y r a t e f o r mucous g l y c o p r o t e i n s from human t r a c h e a l exp l a n t s i n our hands has demonstrated a s i m i l a r s e c r e t o r y response to cholinomimetic agents f o r CF and n o n - b r o n c h i t i c t r a c h e a l exp l a n t s (26). T h e r e f o r e , a strong case cannot be made f o r i n creased i n t r i n s i c responsiveness to s t i m u l a t i o n of CF mucus s e c r e t i n g elements. D e f i n i t i v e data are not a v a i l a b l e to support the hypothesis that c h o l i n e r g i c tone i s increased i n C F , r e s u l t ing i n a chronic hypersecretory s t a t e ( 2 ) . Abnormal M i l i e u f o r Mucous G l y c o p r o t e i n s : Mucous s e c r e t i o n s of the CF t r a c h e o b r o n c h i a water content and highe s e c r e t i o n s (29). Meconium from meconium i l e u s p a t i e n t s i s a l s o r e l a t i v e l y dehydrated (29). The volume of p a n c r e a t i c s e c r e t i o n s i s diminished i n n e a r l y a l l CF s u b j e c t s , i n d i c a t i n g a decreased water output. The volume of s a l i v a r y s e c r e t i o n s i s not d i m i n i s h ed, but the p r o t e i n c o n c e n t r a t i o n of submaxillary s a l i v a i s higher than normal (29). In s h o r t , mucous-type exocrine s e c r e t i o n s are r e l a t i v e l y dehydrated i n c y s t i c f i b r o s i s . A paucity of water i n these s e c r e t i o n s may e x p l a i n the i n s p i s s a t i o n and d u c t a l or small airways o b s t r u c t i o n seen prominently i n C F . In the lungs, a l a c k of water could reduce the volume of p e r i c i l i a r y f l u i d and i n t e r f e r e with c i l i a r y m o t i l i t y ( 1 ) . In a d d i t i o n , r e l a t i v e l y " d r y " s e c r e t i o n s may have sub-optimal v i s c o e l a s t i c p r o p e r t i e s which would i n t e r f e r e w i t h normal c o u p l i n g of mucus to c i l i a r y movement and consequently w i t h mucus clearance from the lungs (30). The non-mucous s e c r e t i o n s ( p a r o t i d s a l i v a , sweat) of subjects with CF, however, are not reduced i n volume and are not concentrated w i t h respect to organic c o n s t i t u e n t s (29). T h e r e f o r e , a g e n e r a l i z e d defect i n water s e c r e t i o n i s not charac t e r i s t i c of t h i s d i s e a s e . Increased concentrations of calcium are found i n s e v e r a l s e c r e t i o n s of i n d i v i d u a l s w i t h CF, i n c l u d i n g submaxillary and p a r o t i d s a l i v a , t e a r s , and seminal plasma (29). Formation of complexes between d i v a l e n t c a t i o n s and the h i g h l y a c i d i c mucous g l y c o p r o t e i n s may c o n t r i b u t e s u b s t a n t i a l l y to the p h y s i c a l and r h é o l o g i e p r o p e r t i e s of mucous s e c r e t i o n s . For example, r a t goblet c e l l i n t e s t i n a l mucus becomes l e s s s o l u b l e i n the presence of i n c r e a s i n g concentrations of c a l c i u m (31). However, a s i m i l a r e f f e c t i n CF mucus s e c r e t i o n s has not been d e s c r i b e d . F u r t h e r more, calcium-mucin complex formation i s an u n l i k e l y pathogenetic f a c t o r because calcium l e v e l s are not elevated i n CF tracheob r o n c h i a l s e c r e t i o n s , c e r v i c a l s e c r e t i o n s , and meconium (29). The m i l k y appearance of CF submaxillary s a l i v a i n i t i a l l y was
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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considered to be evidence f o r c a l c i u m - g l y c o p r o t e i n i n t e r a c t i o n s (32) but the i n s o l u b l e m a t e r i a l has been shown to be a complex of calcium and a low molecular weight phosphoglycoprot e i n , u n r e l a t e d to the mucous g l y c o p r o t e i n s (33). I n t e r a c t i o n s of mucous g l y c o p r o t e i n s w i t h other c a t i o n i c compounds i n s e c r e t i o n s such as the polyamines and t h e i r metab o l i t e s (29), lysozyme, l a c t o f e r r i n , p r o l i n e - r i c h p o l y p e p t i d e s (34) and immunoglobulins (35) may w e l l o c c u r . However, the r o l e which complexes of mucous g l y c o p r o t e i n s and these substances p l a y i n determining the p r o p e r t i e s of mucus has not been f u l l y e x p l o r e d . The p o s s i b i l i t y that these complexes c o n t r i b u t e to a b n o r m a l i t i e s of mucus i n CF deserves c o n s i d e r a t i o n . A l t e r e d Physicochemical P r o p e r t i e s of Mucous G l y c o p r o t e i n s : Mucous g l y c o p r o t e i n s are r e s p o n s i b l e i n l a r g e part f o r the v i s c o e l a s t i c p r o p e r t i e s of t r a c h e o b r o n c h i a l s e c r e t i o n s . Determinants of these p r o p e r t i e s i n c l u d of non-covalent i n t e r a c t i o n gated g l y c o p r o t e i n molecules, r e s u l t i n g i n the formation of a gel matrix. P a r t i c i p a t i o n of other p r o t e i n s i n the formation of t h i s matrix has been suggested (36). Human r e s p i r a t o r y t r a c t mucous g l y c o p r o t e i n s c o n t a i n 75-80% carbohydrate, 20% or l e s s p e p t i d e , and a v a r i a b l e amount of s u l f a t e e s t e r (40), at l e a s t some of which i s present as D-galactose-6-S0^ (41). Fucose, g a l a c t o s e , glucosamine, galactosamine, and N-acetylneuraminic a c i d are the only i d e n t i f i a b l e sugar r e s i d u e s and are arranged i n a v e r y heterogeneous group (41) of galactosaminyl-threonine ( s e r i n e ) - l i n k e d o l i g o s a c c h a r i d e c h a i n s . Nearly one-half of the peptide amino a c i d s are threonine or s e r i n e r e s i d u e s , and at l e a s t 50% of these hydroxyamino a c i d s are g l y c o s y l a t e d (42). The molecular weight of reduced and carboxymethylated r e s p i r a t o r y g l y c o p r o t e i n prepared from lung washings of an i n d i v i d u a l with c y s t i c f i b r o s i s i s approximately 250,000 as determined by sedimentation e q u i l i b r i u m s t u d i e s (42), but these g l y c o p r o t e i n s behave as i f they are much l a r g e r molecules on agarose g e l f i l t r a t i o n (40). In the non-reduced s t a t e they p o l y merize to form complexes with estimated molecular weights of 600,000 to 3,000,000 (35,43). I t has been suggested that r e p u l s i v e forces between s i a l i c a c i d c a r b o x y l and s u l f a t e a n i o n i c s i t e s on adjacent o l i g o s a c c h a r i d e chains are r e s p o n s i b l e f o r the extended conformation of these g l y c o p r o t e i n s (44), but the cont r i b u t i o n of a n i o n i c groups to the r h é o l o g i e behavior of mucous g l y c o p r o t e i n s c u r r e n t l y i s a subject of debate (45,46). We have developed a system f o r p u r i f i c a t i o n of mucous g l y c o p r o t e i n s i n sputum or lung lavage f l u i d s and t h e i r f r a c t i o n a t i o n based on charge (40). Reduced and carboxymethylated g l y c o p r o t e i n s c o n t a i n i n g a l l of the blood group substance, are r e c o v ered at the v o i d volume of a B i o G e l A-5m column and separated i n t o a s e r i e s of components, ranging from s p a r s e l y a c i d i c to h i g h l y a c i d i c , on DEAE c e l l u l o s e (Figure 2 ) . These components have s i m i l a r peptide and carbohydrate compositions. The major
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
PHOSPHATE
PHOSPHATE
pH 8.0
pH 6.0
0 8 -
S
0 6
CVJ
.
0
M
N
o
C
I
Κα)
I.Or
1
2
0.3M N o C 0 2
3
HI
1
0.4
Ο
6
0.2
10
2C 20
30
F R A C T I O N
40
50
60
N U M B E R
Archives of Biochemistry and Biophysics Figure 2. Fractionation of reduced, carboxymethyhted mucous glycoproteins from a tracheostomized subject with no evidence for lower respiratory tract disease on DEAE cellulose (12 X 0.75 cm column). Glycoproteins were first purified by BioGel A-5m chromatography (40,). Glycoproteins were applied to the column in 0.005M phosphate buffer (pH = 8.0) and eluted stepwise as indicated. Five mL fractions were col lected. Less than 1% of material applied to the column was recovered in the run-through volume. Approximately 60% of material applied to the column was recovered in the four fractions. All four fractions con tained blood group-active substance and were typical mucous glycopro teins with respect to sugar and amino acid composition (40).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic
Fibrosis
115
s p a r s e l y a c i d i c component (I) i s a potent blood group a n t i g e n , has r e l a t i v e l y weak i n f l u e n z a v i r u s hemagglutination i n h i b i t i o n p r o p e r t i e s , and contains 1-2% s u l f a t e . The major a c i d i c com ponent (II) i s a l e s s potent blood group a n t i g e n , a stronger i n h i b i t o r of v i r u s hemagglutination, and i s h i g h l y s u l f a t e d (6-7% by w e i g h t ) . O l i g o s a c c h a r i d e chains of component I I have a s l i g h t l y longer mean c h a i n length than those of component I . However, both components d i s p l a y a wide range of o l i g o s a c c h a r i d e chain l e n g t h s , from 1-20 sugar r e s i d u e s / c h a i n . In general s u l fated sugars are found i n the long c h a i n s , and s i a l i c a c i d r e s i d e s i n chains c o n t a i n i n g 4-7 sugar residues (47). C y s t i c f i b r o s i s t r a c h e o b r o n c h i a l mucous g l y c o p r o t e i n s d i f f e r from those of subjects with other hypersecretory s t a t e s , e . g . , chronic b r o n c h i t i s , i n that they c o n t a i n predominently the h i g h l y a c i d i c components as shown i n F i g u r e 3. Consequently, CF t r a c h e o b r o n c h i a l s e c r e t i o n s as a whole are more h i g h l y s u l f a t e d and e x h i b i t stronger a c i d i been made i n our l a b o r a t o r y and by others (48) . CF s a l i v a r y g l y c o p r o t e i n s are more potent i n h i b i t o r s of i n f l u e n z a Β v i r u s hemagglutination than are s a l i v a r y g l y c o p r o t e i n s from c o n t r o l subjects (49), suggesting that they a l s o have increased a c i d i c properties. No unique mucous g l y c o p r o t e i n components have been found i n CF s e c r e t i o n s and none are m i s s i n g . There i s no evidence f o r a l t e r a t i o n s of the fucose and s i a l i c a c i d contents of CF t r a c h e o b r o n c h i a l mucous g l y c o p r o t e i n s , as postulated a number of years ago f o r i n t e s t i n a l g l y c o p r o t e i n s by Dische (50). Expiants of t r a c h e a l e p i t h e l i u m from subjects with CF a l s o s e c r e t e l a r g e amounts of the h i g h l y s u l f a t e d g l y c o p r o t e i n com ponents. T h i s p a t t e r n r e v e r t s to a non-CF type with predomin ance of s p a r s e l y s u l f a t e d g l y c o p r o t e i n (component I) when s e c r e t i o n by expiants i s stimulated by adding methacholine to the c u l t u r e medium during a 6 hour p e r i o d (Table I ) . These r e s u l t s i n d i c a t e that a l t e r a t i o n s i n the d i s t r i b u t i o n of mucous g l y c o p r o t e i n components from CF t r a c h e o b r o n c h i a l e p i t h e l i u m cannot be a t t r i b u t e d to e x c e s s i v e s t i m u l a t i o n through c h o l i n e r g i c mechanisms. The consequences of enhanced s u l f a t i o n of CF r e s p i r a t o r y t r a c t mucous g l y c o p r o t e i n s remain unclear . Evidence that s u l f a t i o n of macromolecules decreases the water of h y d r a t i o n of these substances (51) suggests that increased s u l f a t i o n and a r e l a t i v e p a u c i t y of water i n CF r e s p i r a t o r y t r a c t s e c r e t i o n s are r e l a t e d i n some way. A question of considerable importance i s the r e l a t i o n s h i p between extent of g l y c o p r o t e i n s u l f a t i o n and the genetic defect i n CF. I t has been suggested, based on r e s u l t s of h i s t o c h e m i c a l s t u d i e s , that increased amounts of s u l f a t e d g l y c o p r o t e i n may r e f l e c t long standing i r r i t a t i o n of the lung airways (52). Thus, the increased s u l f a t i o n of CF t r a c h e o b r o n c h i a l g l y c o p r o t e i n s may be secondary to i n f e c t i o u s b r o n c h i t i s . On the other hand, two pieces of information suggest that increased s e c r e t i o n of h i g h l y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
116
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Archives of Biochemistry and Biophysics
Figure 3. Ratios of the amounts of tracheobronchial glycoprotein in sparsely acidic components (I la) to amounts in highly acidic components (II, III) after fractionation on DEAE cellulose (see Figure 2). Secretions for these studies were obtained by bronchial lavage, performed by Robert E. Wood. Glycoproteins were quantitated by determining the total amount of fucose, galactose, galactosamine, glucosamine, and sialic acid in each fraction (40). f
TABLE I D i s t r i b u t i o n of Mucous G l y c o p r o t e i n Components Secreted by Tracheal Expiants of an O-secretor with CF, With and Without Methacholine S t i m u l a t i o n .
No Methacholine I
II
Methacholine'
III
I
II
III
34
49
16
57
32
11
Fucose^
3.3
4.0
—
1.2
0.9
—
Galactose^
6.1
6.3
—
2.7
3.1
—
1.0
1.0
—
1.0
1.0
—
2.6
5.1
—
2.3
3.2
—
0.8
0.3
—
0.3
0,3
—
% of T o t a l
Carbohydrate
2 Galactosamine • 2 Glucosamine N-acetylneuraminic 1. 2. 3.
acid"'"
Analyzed by gas l i q u i d chromatography (40); r e s i d u e s / 1 galactosamine. Analyzed by amino a c i d analyzer (55); r e s i d u e s / 1 galactosamine. 50 ug/ml methacholine added to the c u l t u r e medium for a 6 hour p e r i o d .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous Glycoproteins in Cystic Fibrosis
1.2H
no serum
control serum
S O j
CF serum
Figure 4. Ratio 5 of3 to H in glycopro teins released from rabbit tracheal expiants in the presence of no serum, 50% (v/v) normal serum, and 50% (v/v) CF serum. Data were generated and analyzed as outlined in legend for Figure 1. No serum vs. CF serum; ρ < 0.02. Control serum vs. CF serum; ρ < 0.02. 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
117
118
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
s u l f a t e d g l y c o p r o t e i n s by CF r e s p i r a t o r y e p i t h e l i a may have a c l o s e r r e l a t i o n s h i p to the gene d e f e c t . In double isotope s t u d i e s , the r a t i o of *^S0, to Η - g l u c o s a m i n e incorporated i n t o g l y c o p r o t e i n s secreted by CF r e s p i r a t o r y e p i t h e l i a l expiants i s s i g n i f i c a n t l y greater than the r a t i o f o r g l y c o p r o t e i n s secreted by age-matched c o n t r o l expiants (53). In a d d i t i o n , recent s t u d i e s i n our l a b o r a t o r y (Figure 4) suggest that b r i e f exposure of r a b b i t t r a c h e a l expiants to 50% CF serum e f f e c t s the r e l e a s e of g l y c o p r o t e i n s which are more h i g h l y s u l f a t e d than those r e l e a s e d i n the presence of 50% age and sex-matched c o n t r o l serum, CF t i s s u e s and/or body f l u i d s may possess a p r o p e r t y , independent of c h r o n i c i n f e c t i o n , which modulates the process of g l y c o p r o t e i n s u l f a t i o n , and which may p l a y a r o l e i n the pathogenesis of the lung d i s e a s e . I t i s u n l i k e l y that people with CF are missing a g l y c o p r o t e i n s u l f a t a s e a c t i v i t y , because we have been unable to detect t h i s a c t i v i t y i n both CF and nonCF t i s s u e homogenates an sputum. Substrate f o t o r y mucous g l y c o p r o t e i n , l a b e l e d and harvested i n v i t r o . Both i n t a c t and d e s i a l y l a t e d mucin were t e s t e d . Enzyme a c t i v i t y was assayed at s e v e r a l pHs, with and without d i v a l e n t c a t i o n s , and with and without T r i t o n X-100 (54). Summary: There are s e v e r a l , w e l l documented a l t e r a t i o n s i n the mucous s e c r e t i o n s of i n d i v i d u a l s with CF, any of which alone or i n combination, may i n t e r f e r e with mucous clearance from the lung or from passageways of other organs. I t seems c l e a r t h a t a d d i t i o n a l s t u d i e s to d e f i n e b a s i c aspects of normal mucous b i o c h e m i s t r y , physiology and pharmacology must precede or p a r a l l e l s t u d i e s of CF mucous s e c r e t i o n s i f data p e r t i n e n t to CF pathophysiology are to be generated. P a r t i c u l a r needs i n c l u d e an understanding of the chemical determinants of p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s of these s e c r e t i o n s , and e l u c i d a t i o n of mechanisms f o r c o n t r o l of s u l f a t e d g l y c o p r o t e i n b i o synthesis. Acknowledgements T h i s work was supported i n part by Grants HL 14844 and HR 52957 from the United States P u b l i c H e a l t h S e r v i c e , and a Grant from the C y s t i c F i b r o s i s Foundation of C l e v e l a n d .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5. BOAT AND CHENG 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Mucous Glycoproteins in Cystic Fibrosis
Literature Cited Wood, R.E., Boat, T.F., and Doershuk, C.F. Am. Rev. Resp. Dis. 113:833 (1976). Davis, P., and di'Sant Agnese, P.A. New Eng. J. Med. 295:481 (1976). Mayo, B.J., Klebe, R.J., Lankford, B.J., Morris, N.R., Barnett, D.R., and Bowman, B.H. Texas Reports on Biology and Medicine 34:107 (1976). Conod, E.J., Conover, J., and Gaerlan, P. Pediat. Res. 11:45 (1977). Mangos, J.Α., and McSherry, N.R. Pediat. Res. 2:378 (1968). Dearborn, D.G. In: Advances in Polyamine Research, Vol. II, Eds. D. Morris, R. Campbell, D. Daves, D. Bartos, and F. Bartos, Raven Press, New York (1978), pp. 273-279. Bowman, B.H., Lankford B.J. Fuller G.M. Carson S.D. Kurosky, Α., and Commun. 64:1310 (1975) Epstein, J., and Breslow, J.L. Proc. Natl. Acad. Sci. USA, 74:1676 (1977). Rao, G.J.S., and Nadler, H.L. Pediat. Res. 9:739 (1975). Shapira, E., Martin, C.L., and Nadler, H.L. J. Biol. Chem. 252:7923 (1977). Davis, S.S. Bull. Physio-path. Resp. 9:47 (1973). Potter, J.L., Spector, S., Matthews, L., and Lemm, J. Am. Rev. Resp. Dis. 99:909 (1969). Khan, M.A., Wolf, D.P., and Litt, M. Biochim. Biophys. Acta 444:369 (1976). Reid, L., and deHaller, R. Mod. Probl. Pediat. 10:195 (1967). Zuelzer,W.W.,and Newton, W.A. Pediatrics 4:53 (1949). Reid, L. In: Ciba Foundation Study Group No. 32, Cystic Fibrosis, Eds. R. Porter and M. O'Connor, Little, Brown and Co., Boston (1968), pp.45-67. Oppenheimer, E.H., and Esterly, N.R. Arch. Path. 96:149 (1973). Bowman, B.H., Lockhart, L.H., et al. Science 164:325 (1969). Bogart, B.I., Conod, E.J., and Conover, J.H. Pediat. Res. 11:131 (1977). Czegledy-Nagy, E., and Sturgess, J.M. Lab. Invest. 35:58 (1976). Boat, T.F., Kleinerman, J.I., Carlson, D.M., Maloney, W.H., and Matthews, L.W. Am. Rev. Resp. Dis. 110:428 (1974). Baker, A.P. "GAP" Conference Reports, Cystic Fibrosis Foundation (1976). Boat, T.F., Cheng, P.W., and Wood, R.E. Mod. Probl. Pediat. 19:141 (1977). Baker, A.P., Hillegass, L.M., et al. Am. Rev. Resp. Dis. 115:811 (1977).
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25. Baker, A.P., and Hillegass, L.M. Arch. Biochem. Biophys. 165:597 (1974). 26. Boat, T.F., and Cheng, P.W. In: Cystic Fibrosis, Projec tions into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Corp., New York, 1976, pp. 165-177. 27. Neutra, Μ., Grand, R.J., and Trier, J.S. Lab. Invest. 36:535 (1977). 28. Sturgess, J., and Reid, L. Clin. Sci. 43:533 (1972). 29. Dearborn, D.G. In: Cystic Fibrosis: Projections into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Co., New York, 1976, pp. 179-191. 30. Wood, R.E., Wanner, Α., Hirsch, J., and Farrell, P.M. Am. Rev. Resp. Dis. 111:233 (1975). 31. Forstner, J.F., and Forstner G.G Pediat Res 10:609 (1976). 32. Gugler, E., Pallavicini, , , , Agnese, P.A. J. Pediat. 71:585 (1967). 33. Boat, T.F., Wiesman, U.N., and Pallavicini, J.C. Pediat. Res. 8:531 (1974). 34. Bailleul, V., Richet, C., Hayem, Α., and Degand, P. Clin. Chim. Acta 74:115 (1977). 35. Degand, P., Roussel, P., Lamblin, G., Durand, G., and Havez, R. Bull. Physio-Path. Resp. 9:199 (1973). 36. Roberts, G.P. Arch. Biochim. Biophys. 173:528 (1976). 37. Shih, C.K., Litt, M., Khan, M.A., and Wolf, D.P. Am. Rev. Resp. Dis. 115:989 (1977). 38. Snary, D., Allen, Α., and Pain, P.H. Biochem. Biophys. Res. Commun. 40:844 (1970). 39. Hill, H.D., Reynolds, J.A., and Hill, R.L. J. Biol. Chem. 252:3791 (1977). 40. Boat, T.F., Cheng, P.W., Iyer, R., Carlson, D.M., and Polony, I. Arch. Biochem. Biophys. 177:95 (1977). 41. Roussel, P., Lamblin, G., Degand, P., Walker-Nasir, E., and Jeanloz, R.W. J. Biol. Chem. 250:2114 (1975). 42. Cheng, P.W., and Boat, T.F. Fed. Proc. 35:1444 (1976). 43. Lopez-Vidriero, M.T., Das, I., and Reid, L.M. In: Respira tory Defense Mechanisms, Part I, Eds., J.D. Brain, D.F. Proctor, and L.M. Reid. Marcel Dekker, New York, (1977), pp. 289-356. 44. Gottschalk, Α., and Thomas, M.A.W. Biochim. Biophys. Acta 46:91 (1961). 45. Meyer, F.A., King, M., and Gelman, R.A. Biochim. Biophys. Acta 392:223 (1975). 46. Deman, J., Mareel, Μ., and Brunyneel, E. Biochim. Biophys. Acta 297:486 (1973). 47. Cheng, P.W. Unpublished Observation. 48. Lamblin, G., Lhermitte, M., Lafitte, J.J., Filliat, M., Degand, P., and Roussel, P. Bull. Europ. Physiopath. Resp.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic Fibrosis
121
13:175 (1977). 49. Boat, T.F., Davis, J., Cheng, P.W., and Stern, R.C. Proceed ings of the Fourth International Symposium on Glycoconjugates, In press. 50. Dische, Z., di Sant'Agnese, P.Α., Pallavicini, C, and Youlos, J. Pediat. 24:74 (1959). 51. Ablett, S., Lillford, P.J., Baghdadi, S., and Derbyshire, W., In: Magnetic Resonance in Colloid and Interface Science. Eds., H. Resing, and C. Wade, A.C.S. Symposium Series 34 (1976), p. 344. 52. Jeffery, P.K., and Reid, L.M., In: Respiratory Defense Mechanisms, Part I, Eds., J.D. Brain, D.F. Proctor, and L.M. Reid, Marcel Dekker, New York, (1977), pp. 193-245. 53. Boat, T.F., and Cheng, P.W., In: Cystic Fibrosis: Projections into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Corporation New York (1976) pp. 165-177. 54. Jentoft, N., and Cheng, Unpublishe 55. Cheng, P.W., and Boat, T.F. Anal. Biochem. 85:276 (1978). 56. Boat, T.F., and Kleinerman, J.I. Chest 67:32 S (1975). RECEIVED April 17, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6 Glycoprotein Storage Disease J. NEVIN ISENBERG Pediatric Gastroenterology Division, Department of Pediatrics—UCLA, Center for the Health Sciences, Los Angeles, CA 90024
I. Alpha-1-antitrypsi attracted considerable attentio cause of its association with two divergent disease processes emphysema and cirrhosis. On routine serum protein electrophor esis, it is the predominant protein in the α globulin area. Iron ically (with regard to the current topic), the disease states are associated with a marked reduction in the protein appearing in this band. Thus, this routine clinical test can serve as a screening test for alpha-1-antitrypsin deficiency-type emphysema or cirrhosis. This subject has been reviewed in 1976 by Harvey L. Sharp, M.D., who first recognized the association between α1AT deficiency and childhood cirrhosis.1 In the subsequent paragraphs, we will utilize the material presented there and update the cur rent status with information published since then. 1
The protein has a molecular weight in the range of 50,000. It has approximately 12% carbohydrate which includes galactose, mannose, N-acetyl-glucosamine and sialic acid. The name, α1AT, comes from its electrophoretic position (-α1-) and from the fact that it is responsible for 90% of the serum trypsin inhibitory capacity. Since this potential may be less important physiologi cally than the inhibitory capacity for other proteases, it has also been referred to as α1 protease inhibitor as the study of protease inhibitors (P ) has increased in general. As it is an acute phase reactant, its serum levels and presumably synthesis increase during inflammation. i
Serum contains about 2ragof α 1AT per ml which has the capacity to inhibit 1.1 mg of trypsin. Both quantity (immuno diffusion methods) and function (trypsin inhibitory capacity) are used to evaluate serum from patients with emphysema or cir rhosis. The complexities begin when the deficient serum is typed. Using an acid starch gel electrophoresis,Fagerhol first appreciated the microheterogeneity of alAT which results in three major and five minor protein bands as products of each 0-8412-0452-7/78/47-080-122$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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allele.2 When this separation was followed by antigen-antibody crossed electrophoresis, the f u l l range of 24 phenotypes for this genetic locus was appreciated. Since each a l l e l e i s expressed by a gene product (with microheterogeneity) the inheritance pattern i s regarded as autosomal codominant. P^ type "M" i s the predominant phenotype and has moderate ëLectrophoretic mobility. Phenotypes moving more rapidly are identified by letters which are before "M," while slower ones are designated by letters after "M." Reduced circulating alAT has been associated with a l l e l e s Ζ ( < 10%), Ρ (12.5%), S (30%) and with P j — i i which no gene product has been identified. The "M" a l l e l e i s 50% (MM=100%). Disease states are associated, then, with diploid phenotypes giving low a c t i v i t y such as PiZZ, PjSZ, Pj — , etc. Population genetic studies have shown the wide variation i n a l l e l e distribution. The highest gene frequency for P±Z i s .0262. i n Swedes vs. .011 pathologic a l l e l e most Pathologic P-^ types are very infrequent among non-whites. The Pj^ a l l e l e system seems closely linked to the locus (IgG heavy chain marker) with placement on chromosome 2,8 or 12 l i k e l y . — In Sveger's study2, 120 PjZZ infants and 48 PjSZ infants were de tected fran 200,000 l i v e births, indicating that one infant i n 1200 births had a potentially pathologic phenotype! The figure (proportionately lower) for the USA makes this inherited glyco protein disorder among the most conmon genetic disorders. Pre natal diagnosis i s not possible. The Lung Disease The postulated mechanism by which emphysema develops i n the deficient phenotypes i s thought to be the result of uninhibited proteolysis that destroj^s the fibrous network of the lungs. alAT does inhibit the leukocyte proteases including elastase and c o l lagenase and i s small enough to leave the vasculature to diffuse to a s i t e of inflanmation to function as a protease inhibitor. The susceptibility of hétérozygotes (e.g., PjMZ) to environmental influences which would provoke lung damage has been disputed.! In a recent study heterozygous children compared with normals fran the same families showed no differences except i n forced flow pulmonary functions analyzed by matched pairs .iL These abnormalities were of the type seen with <*1AT deficiency emphysema and suggest a predisposition for the hétérozygote. Only followup w i l l c l a r i f y the significance of this finding. The Liver Disease When Sharp f i r s t associated childhood cirrhosis with α 1AT deficiency (PjZZ), the hepatocytes contained eosinophilic cyto plasmic granules that were shown to be PAS positive after diastase
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digestion of glycogen. These globules are most evident in peripor t a l hepatocytes and become more prominent with age i n those c h i l d ren affected. These granules are positive when sections are treated with fluorescein-labelled antibody to otlAT. These glob ules are found on close inspection i n individuals with PjZ pheno type whether or not cirrhosis i s present. A single report has identified globules i n the l i v e r of a 79-year-old man with PjMM phenotype that were PAS(+) and positive for alAT by irrmunofluorescent and iirrnunoperoxidase methodology.^ The significance of alAT globules i n the absence of a pathologic phenotype i s not clear at present. This material appears i n dilated portions of the endoplasmic reticulum (ER) and i n no other organelle when specimens are exam ined with the electron microscope. It i s usually amorphous with a fine granular or occasional f i b r i l l a r appearance Both smooth and rough endoplasmic reticulu apparatus. Cells are no have any material.Ζ These are concentrated i n periportal areas. The globules vary i n size from one to 40 microns i n diameter, with 15-20 microns diameter particles predominating, The l i v e r injury begins at b i r t h as evidenced by the f r e quency of cholestasis i n newborns with P^ZZ or PjSZ phenotypes. 1> 3 However, some children escape this phase but demonstrate cirrhosis when l i v e r biopsy i s done as part of the c l i n i c a l evaluation of hepatosplenomegaly. The cholestatic phase usually passes within six months. Although affected children then remain asymptomatic u n t i l cirrhosis becomes s u f f i c i e n t l y advanced to result i n por t a l hypertension with ascites and/or hematemesis, l i v e r function tests show continued evidence for hepatocellular injury without cholestasis. Interestingly, however, hyperbilirubinemia may pre cede enzyme elevation i n the newborn period! While originally the l i v e r disease presenting i n early c h i l d hood was thought to be almost uniformly f a t a l i n adolescence secondary to the consequences of advancing cirrhosis, others have had experiences to the contrary.§. The Toronto group reported four of 18 patients with neonatal cholestasis who have no l i v e r function or physical abnormality i n mid-adolescence.§ Early c i r rhosis with or without neonatal cholestasis was a bad prognostic factor.?. In Sveger's neonatal screening study, the PjZZ infants were c l a s s i f i e d into three groups according to the severity of hepatic involvement .-2. Fourteen of 122 had prolonged obstructive jaundice, eight had a suspicion of l i v e r disease by examination, and the remainder had no c l i n i c a l evidence for l i v e r disease. The PjSZ children were a l l i n the third category. Continued evalua tion of the infants over their f i r s t s i x months of l i f e showed that half of those i n the third category with P-^ZZ phenotypes had biochemical abnormalities indicating l i v e r injury. This was not
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found i n the SZ group. Sass-Kortask could draw no prognostic significance from b i opsies taken during the cholestatic phase. Ihey saw equivalent degrees of injury i n well survivors and early deaths Λ French workers have foimed a different conclusion on the basis of re viewing biopsies from fifteen P ZZ children with neonatal chole stasis . ϋ Those that shewed improvement i n l i v e r injury had chole stasis and c e l l u l a r injury, but only slight portal fibrosis. When fibrosis was extensive and b i l e ducts proliferated, early cirrho s i s occurred. A t h i r d group, i n which prolonged cholestasis was noted, had ductular hypoplasia. The follow-up period for most of the patients i n the series i s less than five years, which i s i n sufficient to make a f i n a l judgement; but those i n Groups II and III are progressing toward the complications of cirrhosis while those i n Group I seem to be inprovingJ* A l l had dPAS(+) globules whose size and nuirber di grouping.9 i
PAS(+) globules are also noted i n emphysematous adults of Pj^ZZ phenotype..1 Fibrosis, cirrhosis and hepatoma were noted. No alAT globules are seen i n the malignant c e l l s . — Adults with phenotypes resulting i n p a r t i a l deficiencies had no greater i n c i dence of l i v e r disease than PjMM c o n t r o l s . i l The Liver Storage Material Clearly, the accumulation (storage?) of alAT-like material in the l i v e r seems well documented and i s associated with cirrho s i s . The mechanism by which the hepatic injury occurs i s more d i f f i c u l t to envision than that proposed for the development of emphysema i n deficient phenotypes. The l i v e r disease i s not merely a product of deficient serum alAT since i t seems to be most easily related to the PjZ a l l e l e and not other deficient pheno types. Further, the PjZ protein retains f u l l protease inhibitor capacity proportionate to the amount present. An understanding of the mechanism for l i v e r injury w i l l very l i k e l y require a better understanding of how the α lAT-like mat e r i a l gets to the l i v e r i n the f i r s t place. It i s synthesized by the l i v e r and recently has been p a r t i a l l y characterized as a pro duct of human l i v e r c e l l monolayer cultures. PjM and Pj^Z types were cultured. Only the PjZ c e l l s shewed globules with alAT immunofluorescent p o s i t i v i t y . Ihe P.Ζ protein fran the c e l l s d i f fered from that released to the culture medium and extracted from the supernatant.— This may become an important laboratory ap proach to understanding more about the pathogenesis of l i v e r dis ease. In his review, Sharp l i s t e d four hypotheses that might ex plain the accumulation of alAT i n the livers of P-ZZ individuals:
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1) A point mutation leading to an amino acid substitution i n the peptide which would interfere with subsequent transport from the l i v e r ; 2) A glycyl transferase deficiency resulting i n an inade quately glycosylated protein that could not be transported; 3) P^ZZ i s synthesized and secreted but more rapidly taken up by the l i v e r ; and 4) A pro- alAT i s the storage product because a cleaving enzyme i s missing. It could be added that the amino acid substitution (1) might result i n inadequate glycosylation (2) and secretory failure. Many groups are working on isolation of alAT from serum and l i v e r . No one has found a markedly larger alAT i n the l i v e r so that support for pro-alAT i s minimal. Increased hepatic uptake i s a known feature of asialoglycoproteins. Working with purified alAT, Gan's group shewed that removing s i a l i c acid an galactose residue, altere change binding to concanavilin A, trypsin, or chymotrypsin inhib itory activity or immunological reactivity .1!L The unaltered alAT had a h a l f - l i f e of 18 hours. After neuraminidase treatment re moving four of s i x s i a l i c acid residues, the h a l f - l i f e was re duced to 30 minutes. With subsequent treatment of the galactose residues with galactose oxidase, the h a l f - l i f e was restored to 18 hours .12 Similarly, isolated M and Ζ alAT were labelled with radio active iodine and injected into PjMM subjects. Simultaneous dis appearance rate measurement showed the h a l f - l i f e of M equal to seven days and for Ζ five days. Ihese differences are too small to explain the serum deficiency seen i n P^ZZ.!! Many investigators have suggested that the α 1AT of P^ZZ i s deficient i n s i a l i c acid, but the reported amount and composition of the carbohydrate on this variant protein shows many discrepan cies from group to group. In 1975, the Swedish group reported isolation and characterization of the alAT P^Z from the inclusion bodies i n a P^ZZ l i v e r . 15 It contained no s i a l i c acid, was v i r tually insoluble, f a i l e d to inhibit proteases but did shew thesanre irrinunoreactivity as serum alAT.l§_ Its amino acid composition was similar to PjM but the sugars were a l l reduced to the following percent of that found i n PjM: mannose - 50%, galactose - 25%, N-acetylglucosamine - 16%. These findings, i n the investigator's view, suggested a potential substitution i n the amino acid back bone leading to decreased s o l u b i l i t y , ineffective transport through the ER and secondarily inadequate glycosylation. 15. In the same p e r i o d , Sharp joined forces with Glew and co-workers to pursue the latter's o b s e r v a t i o n that sialyltransferase was deficient i n the serum
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of patients with the PjZZ phenotype. After more extensive studies, i t appeared that the decreased serum levels were i n general rela ted to advanced cirrhosis but not accompanied by decreased activ ity i n l i v e r homogenates. No kinetic differences i n the enzyme were noted between the sialyItransferase from PjMM and P±ZZ.± In 1976, two groups reported an amino acid substitution i n the protein backbone of the Ζ variant of alAT. 16,17 Both groups made peptide maps from digests of the isolated alAT made i n two different ways and found differences i n one or more peptides. Both agree that lysine i s substituted for glutamic acid i n the major variant peptide. The USA group believe that another glu tamic acid i n the M peptide i s replaced by glut amine i n the Ζ peptide. Similarly, an amino acid substitution was confirmed i n a histidine containing peptide of the S-variant.l§ Valine was substituted again for glutamic a c i d . T h e S and M variant had the same s i a l i c aci However, dissenting voices have been raised about the Ζ var iant findings. Owen and Carrell could find no peptide fran the Ζ variant in the expected part of the peptide map after t r y p t i c digestion of both M and Z.£0 A sequential analysis of an M pep tide not seen i n the Ζ map enabled prediction where the variant peptide should be. Thus far, sequential analyses of numerous peptides from the digests has f a i l e d to show any amino acid d i f ferences . Further information strengthening the argument for the po t e n t i a l accumulation of an a s i a l o - alAT comes from studies of Bnerson which imply that the l i v e r asialoglycoprotein binding protein (LABP) i s present i n the membranes of the Golgi apparatus and SER. Thus, P-^Z with exposed galactosyl residues secondary to incomplete s i a l y l a t i o n would bind to the SER and be no further t r a n s p o r t e d C o m p a r i n g binding to LABP i n vitro, native serum PiZ binds more avidly than native M but much less well than com pletely desialylated M. Ihis suggests that there i s circulating PjZ which has exposed galactosyl residues. Circular dichroism has been applied to the analysis of alAT fran normal serum. Sixteen to 20% i s α-helix, 30-50% i n the 3 pleated sheet conformation and the remainder i s aperiodic. This conformational p r o f i l e remained stable over a pH range fran 4.7 to 8.8, but was markedly changed by pH 2.5, guanidine hydrochlor ide and SDS. These findings implicate electrostatic interaction, hydrogen bonding and hydrophobic regions as v i t a l to the three dimensional structure of alAT..?? Obviously, the application of this technique to the variant α1AT with single amino acid sub stitutions may y i e l d information on conformational changes that could interfere with glycosylation. Its application to the problem of microheterogeneity w i l l likewise be interesting.
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Microheterogeneity The factors responsible for the microheterogeneity i n the products of a single gene are not resolved. In some glycoproteins the microheterogeneity has been attributed to a variable degree of s i a l y l a t i o n . Using isoelectric focusing to analyze the DEAE c e l lulose - NaCl gradient column chromatographic separation of purif i e d P-^M, five pooled fractions each shaved up to three of the five bands present i n the otiAT preparation before DEAE fractionation. The f i r s t pool eluted with a low NaCl concentration had the more cathodal isoproteins, while the last pool (hi concentration NaCl) had the more anodal ones. If this was due to s i a l y l a tion differences, then Pool X should have been richer i n s i a l i c acid. However, a l l pools were homogenous by irnmunoelectrophores i s and had the same amino acid, hexose, hexosamine and s i a l i c acid concentrations! When the pools were enzymatically desialylated the bands i n each poo and relative positions o This study casts doubt on uneven s i a l y l a t i o n as an explanation for the microheterogeneity i n aiAT.i^L This a b i l i t y to separate the various forms of the gene product w i l l enable the ultimate identification of what i s responsible for the microheterogeneity. Allen has used isoelectric focusing to study the microheterogeneity i n P-^ZZ children with cirrhosis and compared i t to adults with emphysema and PjZZ i n d i viduals with no c l i n i c a l l y evident disease.?â A l l bands inhibit trypsin and were antigenically α 1AT by crossed irnmunoelectrophoresis. The c i r r h o t i c children had higher levels of alAT and increasing amounts with age of isoprotein i n the Z2 (anodal) band region (isoprotein point = 4.534). Ihus, i t appears that there are differences even i n the PiZ product which might be used i n conjunction with newborn screening to predict whether infants of PjZZ phenotype w i l l be at r i s k for l i v e r or lung disease. Using this Z2 band difference as their marker, family studies showed that penetrance of the Ζ a l l e l e was of the order of 70%.25 i f these observations hold true, then i t may be possible to better understand why seme P-^ZZ families have l i v e r disease without lung involvement and why the converse exists. Up to this point, en vironmental factors (which would stimulate the synthesis of acute phase reactant) were being sought to help explain the onset of l i v e r disease i n the newborn. The majority of investigators had been unable to identify any infectious or toxic agent present i n those children developing the cholestatic phase of the disease. 1. Furthermore, since the incidence of small for gestational age i n fants i s increased i n the P^ZZ phenotype, there i s some evidence that the disease may begin i n utero. Therapy No therapy exists for this glycoprotein storage disease.
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Factors that increase serum levels of α 1AT i n P-^MM do not alter the level of P^ZZ. Steroids have not been helpful. Several pat ients have received l i v e r transplants with conversion of serum Pi phenotype from ZZ to MM. A l l died of transplantation compli cations. At autopsy, no PAS(+) globules were noted in the l i v e r which speaks strongly against a peripheral desialylation mechan ism for altering alAT with resultant storage.
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ASPARTYLGLUOOSAMINURIA
In the usual sense, storage disease implies accumulation of a macromolecule whose catabolism i s interrupted by a deficiency in one or more of the hydrolytic enzymes involved i n i t s break down. For glycoproteins, disturbances i n the breakdown of the peptide backbone or carbohydrat accumulation. However a l l proteins and have f a r reaching, perhaps lethal, effects. In addition, glycoprotein accumulation might be the consequence of disturbances i n the breakdown of the carbohydrate side chain. Yet, the carbohydrate side chains are shared by the glycolipids and abnormalities i n their cleavage are discussed elsewhere i n this symposium. The bond unique to the glycoproteins i s that between the f i r s t sugar moiety of the side chain and the amino acid residue in the peptide backbone. In man, the predominant bond i n serum glycoproteins seems to be the N-glycosidic linkage between N-acetylglucosamine and asparagine. The lysosomal enzyme respon sible for the hydrolysis of this bond i s [2-acetamido-l- ( β-Laspartamido) -l,2-dideoxy-3-D-glucose aspartamido hydrolase (EC 3.5.1.26)] AADGase. A deficiency i n this enzyme was f i r s t demonstrated i n two mentally retarded English siblings who had excreted large amounts of an unusual compound i n the urine which was identified as 2-acetamido-l-( 3 -L-aspartamido) -1,2-dideoxy- 3 -D-glucose (AADG).i Ibis was the i n i t i a l recognition of the disorder, asparty lglucosaminuria (AGU), which has subsequently been diagnosed in Finland? and the United States .2 The Finnish workers have demonstrated the prevalence of this disorder i n their country (1:26,000 l i v e births) by screening residents of national institutions for the mentally retarded.4The coarse features, visceromegaly and vacuolated lymphocytes re sult i n c l i n i c a l l y confusing this disease with the mucopolysac charidoses and mucolipidoses.5. Mental retardation characterized by impaired speech and motor clumsiness predominates .2* 4_ Connec tive tissue lesions including dyostosis multiplex, umbilical her nias and hypermobile joints are common.3,4 Systolic heart rauirnurs
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are frequent. Our patient shaved mitral insufficiency on heart catheterization suggesting that this may be another metabolic disorder directly influencing valvular function.! Despite a pre disposition to recurrent infections, no d e f i c i t s i n leukocyte function have been demonstrated Extensive light and electron microscopic studies have been carried out on various tissues from affected patients.§^Z»-§- The major changes i n a l l tissues are lysosomal. In addition to the readily discernible lymphocyte vacuoles on the peripheral smear, electron dense filamentous material i s noted i n the enlarged lysoscmes of leukocytes .ϋ Virtually every lysosome i n the hepa t i c parenchymal c e l l s i s enlarged. Some PAS positive droplets are seen. Ultrastructurally, the lysosomes contain fine granular material, electron dense structures, and electron lucent l i p i d droplets.§^8. Kupffer c e l l lysoscmes are likewise involved.!L§_ Similar lysosomal change of proximal tubule c e l l jejunal enterocytes§, and i n fibroblasts fran a skin biopsy .8 Ihe most s t r i k i n g changes have occurred i n the neuronal cyto plasm from cerebral cortex, cerebellum and thalamic regions.Ζ The cytoplasm i s v i r t u a l l y f i l l e d with membrane bound vacuoles of either electron lucent granular material or electron dense membranous or granular material with occasional l i p i d droplets.No single feature i s pathognomonic for AGU although seldom i s such a variety of lysosomal changes seen i n other storage diseases. The reduced activity of AADGase was f i r s t noted i n a patient by assay of seminal fluid.,! Markedly decreased activity was ob served i n brain, l i v e r and spleen but not i n kidney biopsy mater i a l , â Leukocyte homogenates are also deficient i n AADGase JL Other lysosomal enzymes involved i n the catabolism of the carbohydrate side chain have normal activity levels except for N-acetyl-3-glucosaminidase which i s elevated.*Li* Measurement of the enzyme i n cultured fibroblasts peimits detection of hétérozygotes and enables prenatal diagnosis. 12 AADGase has been isolated from l i v e r of normals and AGU enabling mixing experiments that rule out inhibitors as a cause for the reduced activity. 11 > 1? Only one isoenzyme has been noted. 11»!? The pH optimum ( 7 . 7 ) and ΗΏ ( 0 . 1 6 mM) have been determined on a 1 7 , 5 0 0 f o l d purification of AADGase that was inhibited by N-acetylcysteine but not other SH agents.12 Even from the f i r s t description of AGU, compounds other than AADG were appreciated i n the urine. 1_ Characterization of these plus the nature of the principal tissue storage compound has taken place. The only quantitatively important compound i n t i s sue i s AADG. lâ Ihe higher molecular weight compounds containing asparagine but with additional carbohydrate residues have been termed glycoasparagines and f a l l into neutral and acidic groups when analyzed by high voltage electrophoresis at pH 2 . 0 and pH
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5.3.ϋ Five neutral and nine acidic glycoasparagines were sepa rated and analyzed. Neutral glycoasparagines (N-^ - N 5 ) differed in the number of neutral sugar residues attached to AADG. The acidic glycoasparagines f e l l into three groups with one or two s i a l i c acid residues; the sulphated compounds formed the t h i r d group. The neutral sugars identified were glucosamine > galac tose » mannose > glucose > fucose. Mannose was noted only i n the neutral group, glucose only i n acidic compounds and fucose in both.i^ Aula, working i n conjunction with several Japanese biochemists, has reported the sequence of several glycoasparagines as shown i n the table b e l o w l ^ ^ : GLYOOASPARAGINE
SEQUENCES IN THE URINE OF AGU AADG
a-NANA-(2+3)-3-Gal-(1+4)-AADG ot-NANA- ( 2+4 ) - 3-Gal- ( 1+4)-AADG a-NANA- ( 2+? ) -3-Gal- ( 1+? ) -3-GlcNAc- ( 1+? ) - 3-Gal- ( 1+4 ) -AADG α-Man- ( 1+6 ) - g-Man- ( l+4)-3-GlcNAc-( 1+4)-AADG That these compounds are present i n only small amounts i n the tissues thus f a r studied makes the source of urine glycoasparagines open to speculation. They may have their origin i n connective tissue. We have suggested that the AADG may serve as an accep tor compound for various transferases leading to synthesis of new glycoasparagines which are released from tissue sites after reaching some c r i t i c a l conformâtion.£-
ACKNOWLEDGEMENT To Harvey L. Sharp, M.D., for introducing me to both these diseases of glycoprotein metabolism.
LITERATURE CITED I. Alpha-1-Antitrypsin 1. Sharp HL: Gastroenterology 70: 611 (1976). 2. Fagerhol MK: Scand J Clin Lab Invest 23: 97 (1969).
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LITERATURE CITED (Cont.) I. Alpha-1-Antitrypsin (Cont.) 3. Sveger Τ: Ν Engl J Med 294: 1316 (1976). 4. Ihird International Workshop - Human Gene Mapping: Cytogenetics and Cell Genetics 16: (1976). 5. Vance JC, Hall WJ, Schwartz RH, Hyde RW, Roghmann KJ, and Mudholkar GC: Pediatrics 60 : 263 (1977). 6. Bradfield JWB and Blenkinsopp WK: J Clin Path 30: 263 (1977). 7. Yunis EJ, Agostini Jr RM, and Glew RH: Am J Pathol 82: 265 (1976). 8. Moro SP, Cutz E, CoxOT,Sass-Kortsak A: J Pediatr 88: 19 (1976). 9. Hadchouel M and Gautier M: J Pediatr 89: 211 (1976). 10. Blenkinsopp WK and Haffenden GP: J Clin Path 30: 132 (1977). 11. Fisher RL, Taylor L and Sherlock S: Gastro 71: 646 (1976). 12. Gautier M, Martin JP and Polim G: Biomedicine (Express) 27: 116 (1977). 13. Yu SD and Gan JC: Arch Biochem Biophys 179: 477 (1977). 14. Laurell CB, Nosslin Β and Jeppsson JO: Clin Sci Molec Med 52: 457 (1977). 15. Jeppsson JO, Larsson C and Eriksson S: Ν Engl J Med 293: 576 (1975). 16. Jeppsson JO: FEBS Letters 65: 195 (1976). 17. Yoshida A, Lieberman J, Gaidulus L and Ewing C: PNAS 73: 1324 (1976). 18. Ovens MC, Carrell RW and Brennan SO: BBA 453: 257 (1976). 19. Yoshida A, Ewing C, Wessels M, Lieberman J and Gaidulis L: Am J Hum Genet 29: 233 (1977). 20. Owens MC and Carrell RW: FEBS Letters 79 : 245 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6. ISENBERG Glycoprotein Storage Disease LITERATURE CITED (Cont.) I. Alpha-1-Antitrypsin (Cont.) 21. Emerson WA: GAP Conference 1: 7 (1977). 22. Jirgensons Β: BBA 493 : 352 (1977). 23. Hercz A and Barton M: Can J Biochem 55: 661 (1977). 24. Allen RC and Hennigar GR: Am J Clin Path 67: 209 (1977). 25. Allen RC: GAP Conference 1: 16 (1977). II. ASPARIYLGLUOOSAMINURIA 1. Pollitt RJ, Jenne (1968). 2. Palo J and Mattsson K: J Ment Defic Res 14: 168 (1970). 3. Isenberg JN and Sharp HL: J Pediatr 86: 713 (1975). 4. Autio S: J Ment Defic Res (Monograph Series 1) 1972. 5. Fleisher TA, Isenberg JN and Sharp HL: J Pediatr 87: 833 (1975). 6. Arstila AU, Palo J, Haltia M, Riekkinen Ρ and Autio S: Acta Neuropath (Berl) 20 : 207 (1972). 7. Haltia M, Palo J and Autio S: Acta Neuropath (Berl) 31: 243 (1975). 8. Isenberg JN and Sharp HL: Human Pathol 7: 469 (1976). 9. Palo J, Riekkinen P, Arstila AU, Autio S and Kivimaki T: Acta Neuropath (Berl) &): 217 (1972). 10. Aula P, NäntöV, Laipio ML and Autio S: Clin Gen 4: 297 (1973). 11. Savolainen H: Biochem J 153: 749 (1976). 12. Dugal Β and Strømme J: Biochem J 165: 497 (1977). 13. Palo J, Pollitt RJ, Pretty KM and Savolainen H: Clin Chim Acta 47 : 69 (1973). 14. Pollitt RJ and Pretty KM: Biochem J 141: 141 (1974).
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LITERATURE CITED (Cont.) II. ASPARTYLGLUCOSAMINURIA (Cont.) 15. Sugahara K, Funakoshi S, Funakoshi I, Aula Ρ and Yamashina I: J Biochem 80: 195 (1976). 16. Akasaki M, Sugahara K, Funakoshi I, Aula Ρ and Yamashina I: FEBS Lett 69: 191 (1976). RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7 Lysosomal Enzyme Deficiency Diseases—Glycoprotein Catabolism in Brain Tissue ERIC G. BRUNNGRABER Missouri Institute of Psychiatry and the Departments of Psychiatry and Biochemistry, University of Missouri—Columbia, St. Louis, MO 63139 The heteropolysaccharid chain f glycoprotein i brain tissue provide th substrates for a number of lysosomal glycosidases. The genetically-induced absence of a glycosidase, or a mutation which causes one or more of these glycosidases to become non-functional, produces a block in the catabolism of these proteinlinked heteropolysaccharides. This pathological event, observed in the lysosomal enzyme deficiency diseases, causes an accumulation of undegraded glycopeptides or oligosaccharides, and an excessive urinary excretion of these substances. About 65 percent of the glycoprotein-carbohydrate of brain tissue is released in the form of sialoglycopeptides which contain NeuNAc, Fuc, GlcNAc, Gal, and Man (1) after treatment of the glycoproteins with proteolytic enzymes, usually papain or pronase. Approximately 20-40 percent of these sialoglycopeptides contain, in addition, sulfate ester groups. All of these sialoglycopeptides appear to contain 3 Man residues per glycopeptide molecule (Table I). Two, three, and possibly four chains consisting of -GlcNAc-Gal-NeuNAc (Fuc) are attached to this trimannoside core. The sialoglycopeptides were partially resolved by column electrophoresis. Fraction I contains the sialoglycopeptide with the highest molecular size and negative charge. The molecular size TABLE I SIALOGLYCOPEPTIDES DERIVED FROM BRAIN GLYCOPROTEINS Fraction: I II III IV V VI VII NeuNAc 4 4 3 2 1 1 3 Gal 4 4 4 3 3 2 2 Man 3 3 3 3 3 3 3 GlcNAc 6 6 6 6 5 4 4 Fuc 1 1 1 1 2 1-2 1-2 0-8412-0452-7/78/47-080-135$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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and charge decreased as the number and s i z e of the - G l c N A c - G a l NeuNAc (or Fuc) chains attached to the trimannoside core i s r e duced. The composition of f r a c t i o n s VI and VII i s of p a r t i c u l a r interest. These s i a l o g l y c o p e p t i d e s account for about 15-20 p e r cent of the glycopeptide-carbohydrate of whole r a t b r a i n . Two -GlcNAc-Gal-NeuNAc (or Fuc) chains are attached to the trimanno side core. The Man residues are s u f f i c i e n t l y exposed so that these glycopeptides show a greater a f f i n i t y to Con A-Sepharose columns (2) than the more a c i d i c p o l y s i a l o g l y c o p e p t i d e s of f r a c t i o n s I to IV. These glycopeptides a l s o have a greater a b i l i t y to i n h i b i t the p r e c i p i t a t i o n of glycogen by Concanavalin A than the more n e g a t i v e l y charged p o l y s i a l o g l y c o p e p t i d e s (3). I t was a l s o found (4) that these glycopeptides y i e l d m e t h y l a t i o n p r o ducts that resemble those obtained from a glycopeptide from transferrin:
( A)
Gal+GlcNAc (1+2 ) Man (1+6 )
^ Man+Gl cN Ac+Gl cN Ac-*As η Gal+GlcNAc (1+2 ) Man (1+3 ) '
S i a l o g l y c o p e p t i d e s of f r a c t i o n s I to V , c h a r a c t e r i z e d by higher values f o r the r a t i o s of NeuNAc/Man, Gal/Man, and GlcNAc/ Man may possess s t r u c t u r e s which resemble those of the g l y c o peptides d e r i v e d from f e t u i n ( 4 ) , as suggested some years ago (5) and f o r which Krusius et a l C4,6) have obtained some e v i d e n c e . : Gal-GlcNAc^ Man Gal-GlcNAc (B)
\
x
Gal-GlcNAc^ Gal-GlcNAc'"
Man'
/
Man-GlcNAc- Gl cN Ac -Agn
The b r a i n s i a l o g l y c o p e p t i d e s contained r e l a t i v e l y high amounts of t e r m i n a l nonsubstituted G a l and GlcNAc residues which suggested that the p e r i p h e r a l chains are often incomplete (6). As we s h a l l see, an accumulation of glycopeptides with s t r u c t u r e s corresponding to (A) have been shown to accumulate i n f u c o s i d o s i s , I - c e l l d i s e a s e , and the GM1- and GM2-gangliosidoses. An accumulation of o l i g o s a c c h a r i d e s or glycopeptides with 3 or 4 t e r m i n a l -GlcNAc-Gal-NeuNAc (or fucose) branches has not yet been r e p o r t e d . Does t h i s suggest the presence i n t i s s u e s of a h i t h e r t o undescribed endo-g-glucosaminidase which s p l i t s the bond between the GlcNAc r e s i d u e of the -GlcNAc-Gal-NeuNAc(or Fuc) branch chain l i n k e d to the trimannoside core? The existance of such an enzyme might e x p l a i n the r e l a t i v e l y l a r g e accumulation of o l i g o s a c c h a r i d e s and glycopeptides with s t r u c t u r e (A) i n the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
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137
l i v e r , b r a i n and u r i n e i n s e v e r a l of the lysosomal enzyme d e f i ciency d i s e a s e s , s i n c e t h i s s t r u c t u r e would be the accumulated end product of the catabolism of a wide v a r i e t y of h e t e r o p o l y saccharide c h a i n s . Finne et a l (7) demonstrated the presence of d i s i a l o s y l (a-N-acetylneuraminyl-(2+8)N-acetylneuraminyl) groups i n the s i a l o g l y c o p e p t i d e s from b r a i n . Thus, glycopeptides of f r a c t i o n s I and I I , which contain 4 NeuNAc r e s i d u e s , may c o n t a i n d i s i a l o s y l groups, ensuring the a v a i l a b i l i t y of one or two of the 4 Gal residues as a t e r m i n a l sugar or as a sugar that i s p e n u l t i mate to a t e r m i n a l fucose or s u l f a t e r e s i d u e . Approximately 20-25 percent of the g l y c o p r o t e i n - c a r b o h y d r a t e of r a t b r a i n c o n s i s t e d of mannoglycopeptides which c o n t a i n 6 Man and 2 GlcNAc residues per glycopeptide molecule (8). Manno glycopeptides , r e a d i l y i s o l a t e d since they bind s t r o n g l y to Con A-Sepharose, contained t e r m i n a l Man residues and would not be a f f e c t e d i n the G M l - g a n g l i o s i d o s i would make a major c o n t r i b u t i o n to the accumulated o l i g o s a c c h a r i d e s i n mannosidosis. When p u r i f i e d l i v e r lysosomes were incubated i n the presence of f e t u i n or orosomucoid ( 9 ) , about one h a l f of the peptide bonds were cleaved and most of the t e r m i n a l NeuNAc residues were r e leased. Release of Gal and GlcNAc was slower and d i d not exceed 30 percent of the t h e o r e t i c a l y i e l d . Release of Man was not d e t e c t e d , and branch p o i n t s i n which Man r e s i d u e s were i n v o l v e d appeared to r e s i s t a t t a c k . Many s t u d i e s have shown that p u r i f i e d 3-galactosidase and β - Ν - a c e t y l g l u c o s a m i n i d a s e can cleave t e r m i n a l l y exposed c a r b o hydrate groups of i n t a c t g l y c o p r o t e i n s , and i t appears l i k e l y that t h i s process occurs i n the lysosomes of the i n t a c t organism. There may be an e x c e p t i o n , however. H i g h l y p u r i f i e d a-fucosidase from r a t l i v e r c a t a b o l i z e d the h y d r o l y s i s of 1-2, 1-3, and 1-4 f u c o s y l linkages i n g l y c o p e p t i d e s , but not i n i n t a c t g l y c o p r o t e i n s (10). Since fucose i s i n a t e r m i n a l l y l i n k e d p o s i t i o n , f a i l u r e to cleave i t w i l l prevent the f u r t h e r degradation of any chains terminated by t h i s sugar. An endo-3-N-acetylglucosaminidase which hydrolyzes the d i - N , N - a c e t y l c h i t o b i o s y l bond i n the l i n k a g e r e g i o n i n g l y c o peptides to produce o l i g o s a c c h a r i d e s w i t h GlcNAc i n a reducing p o s i t i o n and Asn-GlcNAc ( F i g . 2) has been demonstrated i n the hen oviduct (11) and methods f o r i t s assay have been described (12). Although most of the carbohydrate can be removed from n a t i v e g l y c o p r o t e i n s by the endo-glucosaminidase, denaturation of the g l y c o p r o t e i n s was necessary to e f f e c t complete renewal (13). The a c t i v i t y of t h i s enzyme accounts f o r the accumulation of o l i g o s a c c h a r i d e s i n some of the lysosomal enzyme d e f i c i e n c y diseases. O l i g o s a c c h a r i d e s may a l s o be produced by the a c t i o n of 4 - L - a s p a r t y l g l y c o s y l a m i n e amidohydrolase, which cleaves the bond between GlcNAc and asparagine (14). The s i z e and s t r u c t u r e of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
the carbohydrate group d i d not appear to be an important f a c t o r i n c o n t r o l l i n g a c t i v i t y , but the presence of amino a c i d s i n a d d i t i o n to the asparagine i n the peptide p o r t i o n of the g l y c o peptide molecule reduced the r a t e of t h i s r e a c t i o n . T h i s enzyme i s not a c t i v e on i n t a c t g l y c o p r o t e i n s (15). Treatment of the d e l i p i d a t e d b r a i n t i s s u e w i t h p r o t e o l y t i c enzymes r e l e a s e d a l l of the heteropolysaccharide chains of the g l y c o p r o t e i n s , along with glycosaminoglycans and n u c l e i c a c i d s . The l a t t e r substances are removed by p r e c i p i t a t i o n with c e t y l p y r i d i n i u m c h l o r i d e . D i a l y s i s of the glycopeptide p r e p a r a t i o n separates the s i a l o g l y c o p e p t i d e s , which are l a r g e l y n o n - d i a l y z a b l e , from the d i a l y z a b l e , Con-A-binding, mannoglycopeptides. In the storage d i s e a s e s , accumulated degradation products d e r i v e d from the s i a l o g l y c o p e p t i d e s w i l l appear i n the d i a l y z a b l e glycopeptide f r a c t i o n . Thus, an i n c r e a s e i n the carbohydrate content of t h i s f r a c t i o n does not n e c e s s a r i l y i n d i c a t e an ac cumulation of the norma i n d i c a t e the appearanc ( u s u a l l y present only i n t r a c e s ) d e r i v e d from the n o n d i a l y z a b l e sialoglycopeptides. Treatment of the d e l i p i d a t e d t i s s u e with p r o t e o l y t i c enzymes has the disadvantage of obscuring the evidence f o r the p o s s i b l e accumulation of glycopeptides or o l i g o s a c c h a r i d e s i n the t i s s u e . These substances can be recovered q u i t e simply by e x t r a c t i n g the t i s s u e with water or aqueous s o l v e n t s . As we s h a l l see, the l a t t e r procedure has the advantage of p r o v i d i n g the accumulated o l i g o s a c c h a r i d e s which have proved to be amenable f o r s e p a r a t i o n and s t r u c t u r a l a n a l y s i s . A thorough study would allow f o r the i s o l a t i o n of these substances p r i o r to s u b j e c t i n g the i n s o l u b l e t i s s u e r e s i d u e to p r o t e o l y s i s . GM2-gangliosidoses ( d e f i c i e n c y of β - h e x o s a m i n i d a s e ) . Tingey (16) reported that the hexosamine l e v e l i n the de l i p i d a t e d b r a i n t i s s u e from Tay-Sachs diseased p a t i e n t s was elevated. Hexosamine-containing substances c o u l d a l s o be ex t r a c t e d from the residue w i t h b o i l i n g water. While only 3-10 percent of the t o t a l b r a i n hexosamine could be e x t r a c t e d from normal b r a i n i n t h i s manner, the amount of the m a t e r i a l extracted from Tay-Sachs b r a i n was s u b s t a n t i a l l y e l e v a t e d . An e l e v a t i o n of protein-bound hexosamine i n Tay-Sachs disease was confirmed by others (17-21). Suzuki (20) found that the aqueous e x t r a c t prepared from the b r a i n of p a t i e n t s with Sandhoff's disease contained elevated l e v e l s of hexosamine while the i n c r e a s e i n hexosamine c o n c e n t r a t i o n of defatted Tay-Sachs b r a i n t i s s u e and i n aqueous e x t r a c t s prepared from the residue was moderate. Glycopeptides were recovered from the d e l i p i d a t e d c e r e b r a l c o r t i c a l t i s s u e by treatment of the m a t e r i a l w i t h papain (22-24). The NeuNAc content of the d i a l y z a b l e and n o n - d i a l y z a b l e g l y c o peptides obtained from Tay-Sachs b r a i n s was normal. Thus, catabolism of these heteropolysaccharide chains d i f f e r e d from that of the g a n g l i o s i d e s ( F i g . 1 ) : cleavage of the NeuNAc residues
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
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Enzyme
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139
was not dependent on the p r i o r removal of hexosamine. The conc e n t r a t i o n and carbohydrate composition of the n o n - d i a l y z a b l e s i a l o g l y c o p e p t i d e s d i d not d i f f e r from normal v a l u e s . On the other hand, a f o u r - f o l d e l e v a t i o n of hexosamine and mannose content i n the d i a l y z a b l e glycopeptides f r a c t i o n was observed i n both Tay-Sachs and Sandhoff's d i s e a s e s . The accumulated d i a l y z a b l e glycopeptides d i d not bind s t r o n g l y to Con A-Sepharose and presumably d i d not contain the t e r m i n a l Man linkages which i n t e r a c t with the l e c t i n . Thus, the accumulated m a t e r i a l d i d not correspond to the mannoglycopept i d e s , also present i n the d i a l y z a b l e glycopeptide p r e p a r a t i o n . Probably breakdown products of the s i a l o g l y c o p e p t i d e s , these accumulated glycopeptides presumably contained t e r m i n a l GlcNAc residues. Oligosaccharides which contained a trimannosyl core and GlcNAc at non-reducing t e r m i n i were i s o l a t e d from the l i v e r of a case with Sandhoff' GlcNAc-containing o l i g o s a c c h a r i d e , or the excessive e x c r e t i o n i n the u r i n e , was not observed i n Tay-Sachs disease (26). The o l i g o s a c c h a r i d e s were i s o l a t e d by homogenizing the l i v e r i n 10 volumes of chloroform-uiethanol-water ( 1 : 2 : 0 . 3 , v / v / v ) . The m a t e r i a l was then p a r t i t i o n e d i n t o an upper aqueous phase which was evaporated to dryness. Gangliosides were extracted from the d r i e d residue by repeated e x t r a c t i o n with dry chloroformmethanol. Glycopeptides were a l s o i s o l a t e d from d e l i p i d a t e d t i s s u e by means of p r o t e o l y s i s with papain. Tsay and Dawson (27) minced b r a i n t i s s u e i n water. The mixture was sonicated and c e n t r i f u g e d , and the water s o l u b l e m a t e r i a l was f r a c t i o n a t e d by g e l f i l t r a t i o n . The s t r u c t u r e of an i s o l a t e d o l i g o s a c c h a r i d e was determined by treatment with the appropriate g l y c o s i d a s e s , periodate o x i d a t i o n , and GLC analysis. Its s t r u c t u r e corresponded to that of the o l i g o saccharide p r e v i o u s l y i s o l a t e d from the l i v e r of Sandhoff p a t i e n t s (25). The amount i s o l a t e d from b r a i n was extremely small compared to the l e v e l s accumulated i n the l i v e r of these patients. GlcNAc (3,1+2) Man (a, 1+3). GlcNAc (3,1+2)Man (a, 1+6)
Man (3,1-^4) GlcNAc
Whether the o l i g o s a c c h a r i d e s i n b r a i n are a product of b r a i n metabolism i s not e n t i r e l y c l e a r . It i s p o s s i b l e that incompletely degraded m a t e r i a l s produced i n the l i v e r and organs can enter the c i r c u l a t i o n from which they may be taken up by brain c e l l s . On the other hand, b r a i n t i s s u e does appear to contain heteropolysaccharide chains (Table I) with a carbohydrate composition which corresponds to that of the accumulated product.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
The d e f i c i e n c y of hexosaminidase A i n Tay-Sachs d i s e a s e , and hexosaminidases A and Β i n Sandhoff f s disease r e s u l t s i n the accumulation of g a n g l i o s i d e GM2 ( F i g . 1) and o l i g o s a c c h a r i d e s ( F i g . 2, r e a c t i o n D) both of which c o n t a i n t e r m i n a l l y l i n k e d β-hexosamine r e s i d u e s . Both hexosaminidases A and Β have been shown to act on t e r m i n a l N - a c e t y l glucosamines of o l i g o s a c c h a r i d e s derived from g l y c o p r o t e i n s (28). G M l - g a n g l i o s i d o s i s , d e f i c i e n c y of F - g a l a c t o s i d a s e . An o l i g o s a c c h a r i d e c o n s i s t i n g of a degradation product d e r i v e d from g l y c o p r o t e i n s was i s o l a t e d from the l i v e r of p a t i e n t s with G M l - g a n g l i o s i d o s i s (29): Gal(β,1+4)GlcNAc(β, 1+2)Man(α,1+3)^ Gal (β, 1+4) GlcNAc (β, 1+2 )Man (α, 1+6 )
'
Man(β,1+4)GlcNAc
The n o n d i a l y z a b l e a f t e r d i g e s t i o n of the d e l i p i d a t e d residue from the c e r e b r a l gray matter showed an over two-fold e l e v a t i o n i n Gal content (31,32). Man and GlcNAc were a l s o e l e v a t e d . These r e s u l t s suggested the accumulation of glycopeptides with exposed Gal residues. Degradation can proceed no f u r t h e r a f t e r the removal of the t e r m i n a l NeuNAc and fucose r e s i d u e s . There was an es p e c i a l l y marked accumulation of glycopeptides which were d e r i v e d from the NeuNAc-rich, h i g h l y n e g a t i v e l y charged, glycopeptides the sugar content of which was c h a r a c t e r i z e d by h i g h values f o r the Gal/Man and GlcNAc/Man r a t i o s ( f r a c t i o n s I and I I , Table I ) . In a d d i t i o n to these changes, e l e c t r o p h o r e t i c a n a l y s i s of the n o n - d i a l y z a b l e glycopeptide p r e p a r a t i o n suggested that a l a r g e p r o p o r t i o n of the s i a l o g l y c o p e p t i d e s were d e f i c i e n t i n -GlcNAcGal-NeuNAc branches which are attached to the trimannoside c o r e . This change cannot be a t t r i b u t e d to the absence of β - g a l a c t o s i dase, and may represent secondary changes due to the e f f e c t s of the d i s e a s e . In a second case, the c o n c e n t r a t i o n of d i a l y z a b l e glycopeptides c o n t a i n i n g hexosamine, Gal and Man showed a 3-7 f o l d e l e v a t i o n which was dependent on the b r a i n area s t u d i e d . The accumulated glycopeptides d i d not bind to Concanavalin A Sepharose (24). A s i x y e a r - o l d boy was reported by P a t e l et a l (33) to have a 2.5 f o l d e l e v a t i o n i n g l y c o p r o t e i n - g a l a c t o s e i n brain. Tsay and Dawson (27) recovered an o l i g o s a c c h a r i d e from the b r a i n of G M l - g a n g l i o s i d o s i s p a t i e n t s the s t r u c t u r e of which was i d e n t i c a l to that recovered from l i v e r and u r i n e by Wolfe et a l (29,30,31). I t s s t r u c t u r e resembled that of the o l i g o s a c c h a r i d e s recovered from the b r a i n of Sandhoff p a t i e n t s , except for the two t e r m i n a l l y - l i n k e d Gal residues ( F i g . 2 ) . Only a r e l a t i v e l y small amount of t h i s m a t e r i a l was recovered from b r a i n ; the accumulation of g a n g l i o s i d e GM1 and glycopeptides released by p r o t e o l y s i s was c o n s i d e r a b l y g r e a t e r .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Figure
G M 3
GM2
GM1
^-glucosidase
galactosidase
1. Block in ganglioside
$
neuraminidase
^hexosaminidase
/ί-galactosidase
BLOCKED
+ Glc
GM2
Gal
GM1
BLOCKED
IN
DISEASE
GM2-gangliosidosis, and
GAUCHERS
>-Gal
+ Neu Ν A c
GANGLIOSIDOSIS
GANGLIOSIDOSIS
Gal
+ Gal Ν Ac
IN
+
IN
catabolism in GM-l-gangliosidosis, Gauchers Disease
Cer
Cer-GIc
Cer-Glc-Gal
NeuNAc
J 1 i— I
Cer-Glc-Gal
-BLOCKED
— I
-NeuNAc
I
Cer-Glc-Gal-GalNAc
•
2)NeuNAc
1
C e r ( 1 « — 1 . β ) G l c ( 4 « - 1 . β) G a l ( 4 « - 1 , ^ g ) G a l Ν A c ( 3 * - 1 . β)
S «s:
Ci
Ο > w Η
142
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Fuc F u c - G a l - GlcNAc - M a n
1
M a n - G l c N A c —GlcNAc - A s n Fuc-Gal - GlcNAc-Man A
^ (Endo-0-N-glucosaminidase)
^
Fuc-Gal-GlcNAc-Man
Fuc \ Man —GlcNAc
I GlcNAc-Asn
+
Fuc- Gal-GlcN Ac-Man Β Gal — G l c N A c —Man ΓM a n — G l c N A c
+
Fuc
Gal — G l c N A c — M a n ' C
\
G l c N A c - A s n + Fuc N-aspartyl-
^
glucosaminidase
( β-Galactosidase)
GlcNAc
+ Asn
GlcNAc — Man
\
G l c N A c —Man
/
Man —GlcNAc
+ Gal
( β-hexosaminidase)
i
Man, Man
>
Man
(Q£
1
-GlcNAc
Man
GlcNAc
mannosidase)
I an —GlcNAc M
(β
+
+ Man
mannosidase) +
GlcNAc
Figure 2. Probable catabolic pathway for the degradation of oligosaccharide chains derived from brain glycoproteins. Reactions B, C, D, and Ε are blocked in fucosidosis, GMl-gangliosidosis, GM2-gangliosidosis, and mannosidosis, respec tively.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
Lysosomal
Enzyme
Deficiency
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Gaucher*s Disease ( d e f i c i e n c y of glucocerebrosidase) Whether p a r t i a l degradation products derived from g l y c o p r o t e i n s accumulate i n the b r a i n of the neuropathic forms of Gaucher s disease i s not known. Kanfer et a l (34) t r e a t e d de f a t t e d l i v e r t i s s u e with p r o t e o l y t i c enzymes. Glycopeptides d e r i v e d from g l y c o p r o t e i n s were markedly elevated i n the Gaucher l i v e r ; a 20 f o l d e l e v a t i o n i n hexose, hexosamine, fucose and NeuNAc was recorded. The accumulated m a t e r i a l contained Gal and GlcNAc as the major sugars; Glc and Man were present i n t r a c e s . The disease i s caused by d e f i c i t i n glucocerebrosidase, a 3-glucosidase. Although glucose-containing g l y c o p r o t e i n s un doubtedly e x i s t (1), there i s no evidence f o r the accumulation of G l c - c o n t a i n i n g glycopeptides or o l i g o s a c c h a r i d e s . Gaucher spleen appears to produce an excess of a g l y c o p r o t e i n a c t i v a t i n g f a c t o r , capable of a c t i v a t i n g the glucocerebrosidase from normal spieen (35-37)· While the a c t i v a t o r from Gaucher spleen was a g l y c o p r o t e i n , that fro composition of the two of the g l y c o p r o t e i n a c t i v a t o r remains obscure. Metachromatic Leukodystrophy (MLD) and M u l t i p l e S u l f a t a s e D e f i c i e n c y (MSP). 1
The s i a l o g l y c o p e p t i d e p r e p a r a t i o n from b r a i n contains mole cules which c a r r y s u l f a t e - e s t e r groups. Consequently, i t ap peared l i k e l y that p a r t i a l degradation products derived from g l y c o p r o t e i n s which bear s u l f a t e d o l i g o s a c c h a r i d e chains might accumulate i n MLD, a disease caused by the absence of a f u n c t i o n ing s u l f a t a s e A. However, s u l f a t i d e s are the n a t u r a l substrate f o r t h i s enzyme. The hexosamine l e v e l s i n the d e l i p i d a t e d t i s s u e residue from cases of MLD, i n which i t was e s t a b l i s h e d that s u l f a t a s e A i s d e f i c i e n t , f e l l w i t h i n the normal range (39,40). The s i t u a t i o n d i f f e r s i n MSD, a v a r i a n t of MLD i n which the t i s s u e s of the p a t i e n t s l a c k s u l f a t a s e s A, B, and C. The non l i p i d hexosamine i n t i s s u e s from such p a t i e n t s show an e l e v a t i o n of protein-bound hexosamine (41), and i t has been shown that t h i s i s due, at l e a s t i n p a r t , by the e l e v a t i o n of glycosaminoglycans· I t i s not known whether glycopeptides or o l i g o s a c c h a r i d e s de r i v e d from g l y c o p r o t e i n s accumulate i n t h i s d i s e a s e . I t has been suggested (42) that O-sulfated glycosaminoglycans are the n a t u r a l substrate f o r s u l f a t a s e B, s i n c e t h i s enzyme i s d e f i c i e n t i n two diseases (MSD and Maroteaux-Lamy disease) i n which O-sulfated glycosaminoglycans accumulate (mainly dermatan s u l f a t e ) . However, c o - c u l t i v a t i o n of MSD f i b r o b l a s t s with S a n f i l i p p o A or Hunter c e l l s d i d not c o r r e c t f o r the degradation defect i n glycosaminoglycan-SO/j-S metabolism. S a n f i l i p p o and Hunter f i b r o b l a s t s l a c k heparan s u l f a t a s e and dermatan s u l f a t a s e , but a r y l s u l f a t a s e Β i n these c e l l s i s normal or elevated (42). These f i n d i n g s l e d to the suggestion that s u l f a t a s e Β may be i n v o l v e d i n degrading s u l f a t e d glycopeptides (43). 35
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S u l f a t a s e Β appears to correspond to N-acetylgalactosamine 4 - s u l f a t a s e (44). The s u l f a t a s e r e s p o n s i b l e f o r the accumula t i o n of protein-bound hexosamine s t i l l remains to be i d e n t i f i e d . Nor i s i t yet e s t a b l i s h e d whether s u l f a t e d glycopeptides d e r i v e d from g l y c o p r o t e i n s accumulate i n MSD. The unknown s u l f a t a s e may be a component of s u l f a t a s e A or B, both of which e x i s t i n m u l t i p l e forms, or a s u l f a t a s e which does not respond to the s y n t h e t i c substrates commonly i n use to detect s u l f a t a s e a c t i " vity. The mucopolysaccharidoses. The defects i n H u r l e r , Hunter, and S a n f i l i p p o A and Β syndromes, those forms of the mucopolysaccharidoses i n v o l v i n g severe n e u r o l o g i c a l d e f i c i t s , are caused by nonfunctioning α - i d u r o n i d a s e , s u l f o i d u r o n a t e s u l f a t a s e , heparan-N-S-sulfatase, and Ofr-N-acetylglucosaminidase, r e s p e c t i v e l y . These linkages a r e , as f a r as i s known, absent i n g l y c o p r o t e i n s . Consequently, no a b e r r a t i o n i n g l y c o p r o t e i such a defect been r e p o r t e d b r a i n of a Hunter p a t i e n t showed no remarkable changes ( 4 5 ) · Diseases with a primary l e s i o n i n g l y c o p r o t e i n c a t a b o l i s m . Mannose-rich o l i g o s a c c h a r i d e s accumulate i n the b r a i n of mannosidosis p a t i e n t s (46-48): Man-Man-Gl cNAc? Man-Man-Man-Gl cNAc Man-Man
^Man-GlcNAc
Man' The disease occurs a l s o i n Angus c a l v e s , and the defatted b r a i n t i s s u e residue of the a f f l i c t e d animals showed a 4-6 f o l d e l e v a t i o n i n Man content (49). D e f i c i e n c y of α - m a n n o s i d a s e causes a b l o c k i n the catabolism of the h e t e r o p o l y s a c c h a r i d e c h a i n s : mannose-containing glycopeptides or o l i g o s a c c h a r i d e s are d e r i v e d from the s i a l o g l y c o p e p t i d e trimannoside c o r e , as w e l l as from the mannoglycopeptides· In p a t i e n t s with a d e f i c i e n c y of ot-fucosidase (fucosidosis), f u c o s e - c o n t a i n i n g substances were shown to accumulate i n the d e l i p i d a t e d t i s s u e , i n c l u d i n g that of the b r a i n (50). Aqueous e x t r a c t s of sonicated b r a i n t i s s u e y i e l d e d an o l i g o s a c c h a r i d e (27) : Fuc-Gal-GlcNAc-Man V
Man-GlcNAc Fuc-Gal-GfccNAc-Man^
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A disaccharide, fucosylated N-acetylglucosamine, was also de tected i n the brains of fucosidosis patients (Fig. 2). Aspartylglucosaminuria i s caused by a d e f i c i t i n the enzyme l-aspartamido-3-N-acetylglucosamine amidohydrolase. Aspartylglucosaminylamine and aspartyloligosaccharides accumulate i n organs, including the brain (51,52). The mucolipidoses are caused by a deficiency of α-neuramini dase (53) resulting i n the excessive excretion of s i a l y l o l i g o saccharides. Most of the oligosaccharides isolated had a core consisting of three mannose residues to which two GlcNAc-GalNeuNAc chains, or incomplete portions of these chains, were attached (54). Although not a l l of the linkages i n the oligo saccharides of brain tissue have been definitively established, i t appears l i k e l y that their structure w i l l prove to be identi cal to that of one of the sialoglycopeptides isolated from urine (55): NeuNAc ( α , 2+6) Gal ( β , 1+4)GlcNA
/
Man(β,1+4)GlcNAc
NeuNAc ( α , 2+6 ) Gal ( 3,1+4 ) GlcNAc ( β , 1+2 )Man (α, 1+6 )' A sequence of the steps involved i n the catabolism of the heteropolysaccharides of brain glycoproteins has been postulated (Fig. 2) based on the structures of the oligosaccharides which accumulate i n the various lysosomal enzyme diseases discussed above. Niemann-Pick's Disease The primary lesion i n the i n f a n t i l e , type A, form of Nie mann Pick's disease i s a deficiency of the enzyme, sphingomye linase. While sphingomyelin i s the primary storage product, many cases which have been examined have demonstrated an increase i n gangliosides and cholesterol i n brain tissue. Tingey (16) and Norman et a l (56) have reported an elevation of proteinbound hexosamine as well. Brunngraber et a l (57) noted a two fold elevation of glycoproteins-carbohydrate which was recovered as sialoglycopeptides upon proteolytic digestion of the d e l i p i dated tissue residue from gray matter. Mannoglycopeptide levels remained within the normal range. The composition of the s i a l o glycopeptides was altered, since the number of -GlcNAc-GalNeuNAc chains attached to the internal trimannoside core was reduced. I t i s possible, but not demonstrated, that the accumu lated glycopeptides i n Niemann-Pick disease may correspond to the sialylated forms of the oligosaccharides which accumulate i n the GMl-gangliosidoses. I t was suggested that sphingomyelin catabolism may be an early step i n the degradation of plasma mem branes, and a lesion i n the catabolism of this phospholipid may have the secondary effect of impeding the degradation of other plasma membrane components.
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Neuronal-Ceroid L i p o f u s c i n o s e s (NCF). A form of NCF may i n v o l v e g l y c o p r o t e i n storage. Adelman et a l (58) found a n e a r l y two f o l d i n c r e a s e i n the NeuNAc con tent of s i a l o g l y c o p e p t i d e p r e p a r a t i o n s obtained from t i s s u e sec t i o n s of the c e r e b r a l c o r t e x of a case with c l i n i c a l f e a t u r e s resembling those of Batten's d i s e a s e . C e l l r e c o g n i t i o n of lysosomal enzymes. Studies u t i l i z i n g c u l t u r e d f i b r o b l a s t s d e r i v e d from p a t i e n t s w i t h lysosomal enzyme d e f i c i e n c y diseases have shown that these c e l l s s e c r e t e lysosomal g l y c o s i d a s e s i n t o the medium, and are capable of r e - a s s i m i l a t i n g these enzymes. Hickman and Neuman (59,60) have proposed t h a t lysosomal enzymes are t r a n s p o r t e d from t h e i r s i t e of s y n t h e s i s and g l y c o s y l a t i o n i n the G o l g i apparatus to the lysosome by t h i s process of e x o c y t o s i s and subsequent p i n o c y t o t i c r e - i n c o r p o r a t i o n . Such a mechanism may be a p a r t of a membrane r e c y c l i n g mechanism s i n c e i n t e r n a l i z a t i o n of the enzyme i s patch of plasma membrane to fuse with the lysosome (61). F i b r o b l a s t s from p a t i e n t s a f f l i c t e d with one of the l y s o s o mal d e f i c i e n c y diseases are capable of a s s i m i l a t i n g the d e f i c i e n t enzyme which had been added to the c u l t u r e medium, thereby c o r r e c t i n g the metabolic d e f e c t . Uptake of lysosomal g l y c o s i dases i s dependent on the r e c o g n i t i o n of the h e t e r o p o l y s a c c h a r i d e c h a i n of the g l y c o p r o t e i n enzyme by a r e c e p t o r on the s u r f a c e of the f i b r o b l a s t . Thus, the t e r m i n a l Gal r e s i d u e appeared to be important f o r the r e c o g n i t i o n and uptake o f a-N-acetylglucosaminidase (62) by f i b r o b l a s t s of S a n f i l i p p o Β p a t i e n t s . Mannose residues appeared to be important f o r uptake of 3-galactosidase by f i b r o b l a s t s from p a t i e n t s with G M l - g a n g l i o s i d o s i s (63). Cultured f i b r o b l a s t s appear to recognize phosphohexosyl groups i n 3-glucuronidase, β-hexosaminidase, and 3-galactosidase (64,65) and a-L-iduronidase (66). The r e c o g n i t i o n of phospho hexosyl groups may be a general c h a r a c t e r i s t i c of p i n o c y t o s i s of lyosomal g l y c o s i d a s e s . These f i n d i n g s may be r e l a t e d to the e a r l i e r r e p o r t s on the i s o l a t i o n of a phosphoglycoprotein from b r a i n (67-69). Phosphorylated mannoglycopeptides were i s o l a t e d from the phosphoglycoprotein, and the phosphate r e s i d u e which i s attached to one of the h y d r o x y l groups of mannose, was removed by the a c t i o n of a l k a l i n e phosphatase (69). The r e l a t i o n of t h i s phosphoglycoprotein to the lysosomal g l y c o s i d a s e s remains to be e s t a b l i s h e d . I f the c a p a c i t y of the f i b r o b l a s t to s e c r e t e and r e - a s s i m i l a t e lysosomal enzymes i s a s p e c i f i c example of a more general phenomena, phosphoglycoproteins and/or g l y c o s i d a s e s may p l a y an important r o l e i n the turnover of the plasma membrane.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Literature Cited Brunngraber, E. G.; Hof, H.; Susz, J.; Brown, B. D.; Aro, Α.; and Chang, I. Biochim Biophys Acta, 304; 781, 1973. Krusius, T. FEBS Lett, 66: 86, 1976. Hof, Η. I.; Susz, J. P.; Javaid, J. I.; and Brunngraber, E. G. Neurobiology, 5.: 347, 1975. Krusius, T.; Finne, J.; and Rauvala, H. FEBS Lett, 71: 117, 1976. Brunngraber, E. G. Adv Exptl Med and Biol, 25: 255, 1972. Krusius, T.; and Finne, J. Eur J Biochem, 78: 369, 1977. Finne, J.; Krusius, T.; and Rauvala, H. Biochem Biophys Res Communs, 74: 405, 1977. Javaid, J. I.; Hof, H. I.; and Brunngraber, E. G. Biochim Biophys Acta, 404: 74, 1975. Aronson, Ν. Ν., Jr.; and DeDuve C J Biol Chem 243: 4564, 1968. Opheim, D. J.; and Touster, 0. J Bio Chem, 252: 739, 1977. Tarentino, A. L.; and Maley, F. J Biol Chem, 251: 6537, 1976. Tarentino, A. L.; and Maley, F. Anal Biochem, 77: 185, 1976. Trimble, R. B.; and Maley, F. Biochem Biophys Res Communs, 78: 935, 1977. Kohno, M.; and Yamashina, I. Biochim Bipphys Acta, 258: 600, 1972. Fidler, I. J.; Montgomery, P.C.;and Cesarini, J. P. Cancer Res, 33: 1376, 1973. Tingey, A. J Neurochem, _3 230, 1959. Stefanko, St.; Guminska, M.; and Pietrzykowa, B. Schweiz Arch Neurol Neurochir Psychiat, 90: 295, 1962. Edgar, G. W. F. Proc of the Fifth Internatl Congr Neuro pathology, Zurich, 1965. Amsterdam, Excerpta Med Found, 1966, p. 328. Thieffry, S.; Bertrand, I.; Bargeton, E.; and Edgar, G.W.F. Rev Neurol, 102: 130, 1960. Suzuki, Y.; Jacob, J.C.;Suzuki,K.;Kutty, Κ.N.;and Suzuki, K. Neurology, 21: 313, 1971. Norman, R. M.; Tingey, A. H.; Newman, C. H. H.; and Ward, S. P. Arch Dis Child, 39: 634, 1964. Brunngraber, E.G.;Haberland,C.;and Brown, B. D. Brain Res, 38: 151, 1972. Brunngraber, E. G.; Brown, B. D.; and Aro, A. J Neurochem, 22: 125, 1974. Brunngraber, E. G.; Davis, L. G.; Javaid, J. I.; and Berra, B. Adv Exptl Med and Biol,68:31, 1976. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 59: 837, 1974. Wolfe, L. S.; and Ng Ying Kin, Ν. M. K. Adv Exptl Med and Biol, 68: 15, 1976. Tsay, G.C.;and Dawson, G. J Neurochem, 27: 733, 1976. American Chemicaf Society Library 1155 16th St. N. W. W a shiGlycolipids ngton, D.inC.Disease 200Processes; 36 In Glycoproteins and Walborg, E.; :
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28. Bearpark, T.; Bouquelet, S.; Fournet, B.; Montreuil, J; Spik, G.; Stirling, J.; Strecker, G. FEBS Lett, 84: 379, 1977. 29. Wolfe, L. S.; Senior, R. G.; and Ng Ying Kin, Ν. M. K. J Biol Chem, 249: 1828, 1974. 30. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 66: 123, 1975. 31. Brunngraber, E. G.; Berra, B.; and Zambotti, V. FEBS Lett, 34: 350, 1973. 32. Berra, B.; Di Palma, S.; and Brunngraber, E. G. Clin Chim Acta, 57: 301, 1974. 33. Patel, V.; Goebel, H. E.; Watanabe, I.; and Zeman, W. Acta Neuropathol, 30: 155, 1974. 34. Kanfer, J. N.; Stein, M.; and Spielvogel, C. Adv Exptl Med and Biol, 19: 225, 1972. 35. Ho, M. W.; and O'Brien J S Proc Natl Acad Sci US 68: 2810, 1971. 36. Ho, M. W.; O'Brien Biochem J, 131; 173, 1973. 37. Ho, M. W. Biochem J, 136: 721, 1973. 38. Peters, S. P.; Coyle, P.; Coffee, C. J.; Glew, R. H.; Kuhlenschmidt, M. S.; Rosenfeld, L.; and Lee, Y. C. J Biol Chem, 252: 563, 1977. 39. Haberland, C.; Brunngraber, E.G.;Witting, L. Α.; and Daniels, A. Acta Neuropathol, 26: 93, 1973. 40. Federico, Α.: Guazzi, G. C.; and Di Benedetta, C. Eur Neurol, 15: 163, 1977. 41. Eto, Y.; Meier, C.; and Herschkowitz, N. N. J Neurochem, 27;1071,1976. 42. Austin, J. Arch Neurol, 28: 258, 1973. 43. Eto, Y.; Wiesmann, U.; and Hierschkowitz, Ν. N. Arch Neurol, 30: 153, 1974. 44. Dorfman, Α.; Arbogast, B.; and Matalon, R. Adv Exptl Med and Biol, 68: 261, 1976. 45. Roukema, P. Α.; Overkerk, C. E. and Van Den Berg, G. Clin Chim Acta, 44: 277, 1973. 46. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; Ockerman, P. Α.; and Autio, S. J Biol Chem, 248: 6210, 1973. 47. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; and Autio, S. Biochemistry, 13: 871, 1974. 48. Tsay, G. C.; Dawson,G.;and Matalon, R. J Pediat, 84: 865, 1974. 49. Norden, Ν. E.; Lundblad, Α.; Ockerman, P. A. and Jolly, R. D. FEBS Lett, 35: 209, 1973. 50. Van Eoff, F.; and Eers, E. G. Eur J Biochem, 7: 34, 1968. 51. Palo, J.; and Savolainen, E. J Chromatog, 65: 447, 1972. 52. Palo, J. and Savolainen, E. Ann Clin Res, 5: 156, 1973. 53. Cantz, M.; Gehler, J.; and Spranger, J. W. Biochem Bipphys Res Communs, 74: 732, 1977.
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54. Michalski, J. C.; Strecker, G.; and Fournet, B. FEBS Lett, 79: 101, 1977. 55. Strecker, G.; Peers, M. C.; Michalski, J. C.; Hondi-Assah, T.; Fournet, B.; Spik, G.; Montreuil, J.; Farriaux, J. P.; Maroteaux, P.; and Durand, P. Eur J Biochem, 75: 391, 1977. 56. Norman, R. M.; Tingey, A. H.; and Fowler, M. C. Proc of the Fifth Internatl Congr Neuropathol, Zurich, 1965, Amster dam, Excerpta Med Found, 1966, p. 143. 57. Brunngraber, E. G.; Berra, B.; and Zambotti, V. Clin Chim Acta, 48: 173, 1973. 58. Adelman, L. S.; Young, E.; and Bass, Ν. H. Neurology, 24: 1045, 1974. 59. Hickman, S.; and Neufeld, E. F. Biochem Biophys Res Communs, 49: 992, 1972. 60. Neufeld, E. F.; Sando, G. N.; Sarvin, A. J.; and Rome, L. H. J Supramol Struct 6: 95 1977 61. Brunngraber, E. G Springfield, Ill. , 62. Von Figura, Κ.; and Kresse, H. Prot Biol Fluids, 22: 275, 1975. 63. Hieber, V.; Distler, J.; Myerowitz, R.; Schmickel, R. D.; and Jourdian, G. W. Biochem Biophys Res Communs, 73: 710, 1966. 64. Kaplan, Α.; Fischer, D.; Achord, D.; and Sly, W. J Clin Invest, 60: 1088, 1977. 65. Kaplan, Α.; Achord, D. T.; and Sly, W. S. Proc Natl Acad Sci US, 74: 2026, 1977. 66. Sando, G. N.; and Neufeld, E. F. Cell, 12: 619, 1977. 67. Davis, L. G.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 30, 1976. 68. Davis, L. G.; Costello, A. J. R.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 35, 1976. 69. Davis, L. G.; Brettschneider, I.; and Brunngraber, E. G. Fed Proc, 36: 750, 1977. RECEIVED
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In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8 Incorporation of Exogenous Enzymes into Lysosomes A Theoretical and Practical Means for Correcting Lysosomal Blockage P. G. PENTCHEV, J.A. BARRANGER, A. E. GAL, F. S. FURBISH, and R. O. BRADY Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20014
The lysosomal storag types for delineating th inherited metabolic disorders. Progress has led to an identification of most primary enzymatic lesions in these disorders and to a better understanding of the cellular manifestations resulting from these enzymopathies. Specific enzymatic tests are now available for the unequivocal diagnosis of most of these storage diseases. Reliable heterozygote detection and prenatal diagnosis has enabled genetic counseling to effectively reduce the incidence of many of these crippling diseases (1). Investigators have also addressed considerable effort to the intriguing possibility of enzyme replacement as an effective therapeutic measure for the treatment of the lysosomal storage disorders. Although more than a decade of effort has been channelled in this direction, an unequivocal answer has yet to appear. An evaluation of the status of this research program can perhaps be best initiated by examining the cellular and biochemical bases for enzyme replacement. Remarkable insight into this phenomena was given in 1964, at a time when only one lysosomal storage disorder was recognized: "Both in our pathogic speculations and in our therapeutic attempts it may be well to keep in mind that any substance which is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction including replacement therapy."(2). The implication of the dynamic nature of lysosomal interactions is that one can, in theory, equate the ability of endogenous and exogenous enzymes to stimulate intralysosomal digestion (Figure I). It is now well documented that enzymes that are initially found in the extracellular environment can be incorporated into the lysosomal architecture of cells (Table I). It has 150 This chapter not subject to U.S. copyright Published 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PENTCHEV ET AL.
Lysosomal
PRIMARY L Y S O S O M E
Blockage
AUT0PHAG0S0ME
ENZ
RESIDUAL B O D Y
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-Λ /
PHAGOSOME
ENDOCYTOSIS
PRIMARY L Y S O S O M E
AUTOPHAGOSOME
r
/
/ •
SECONDARY L Y S O S O M E ,
PHAGOSOME
^ENZ
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ENDOCYTOSIS
Figure 1. Representation of lysosomal interactions. (Top) Nor mal lysosomal digestion. (Bottom) Lysosomal digestion stimuhited by exogenous enzyme.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
also been shown that these "migrant" enzymes are functionally active in reversing previously blocked metabolic pathways and/or reversing abnormal morphology of lysosomes (Table II). In 1966, i t was predicted that Gaucher^ disease might prove to be the ideal model for enzyme replacement therapy (]_5). The accumulation of glycolipid occurs predominantly within the cells of reticuloendethelial system. Consequently intravenously admin istered enzyme preparations were expected to have ready access to major pathological depots of stored l i p i d . Our enzyme infusion studies with Gaucher patients began in 1973 with the successful isolation of human placental glucocerebrosidase (1_6). A brief summary of the results of these i n i t i a l t r i a l s is presented in Table III. Encouraged by these preliminary findings and having developed a more efficient and practical means of purifying large quantities of the enzyme (1_8), we began a series of infusions in patients using considerably more glucocerebrosidase than i n i t i a l l y employed. The results o ing. No significant changes in either the circulating or l i v e r levels of glycolipid were observed following the intravenous ad ministration of native glucocerebrosidase (Brady, R. et al_., un published observations). Concomittant c l i n i c a l observations such as regression of hepatomegaly also prove negative. On the other hand, Belchetz and collaborators (1_9) and Beutler's group (20) have reported a decrease in l i v e r size when glucocerebrosidase was administered over relatively long periods of time (13 and 3-1/2 months respectively). Furthermore, Belchetz, et al_., injected glucocerebrosidase that had been entrapped in liposomes while Beutler, et al^., used resealed erythrocyte ghosts to deliver the placental enzyme. A possible reason for the refractiveness of biochemical and c l i n i c a l responses to exogenous enzyme in our t r i a l s with the new glucocerebrosidase preparations may be an unfavorable cellular disposition of the enzyme. It has been shown that approximately 30% of the infused enzyme is taken up by the l i v e r in rats and monkeys (5). More recent studies have shown that approximately 90% of this uptake represents incorpora tion into hepatocytes ( 2 Π ) . The predominate uptake of enzyme by hepatocytes is an inefficient route for inducing l i p i d clearance since the accumulation of glycolipid occurs mainly within reticuloendothetial cells (22J. Modification and/or encapsulation of the enzymes will now have to be seriously considered in an attempt to specifically target the administered enzyme to these c e l l s . The eventual effectiveness of enzyme supplementation in the treatment of Gaucher's disease will also depend on several other parameters which unfortunately have not been experimentally evalu ated due to the lack of a suitable animal model (Table IV). To begin with, the inherent extent of enzymatically induced clearance will depend on the degree of accessibility of exogenous enzymes to stored l i p i d . Complete clearance of glucocerebroside can be effected when specimens of Gaucher l i v e r tissue are incubated with glucocerebrosidase in vitro (Figure II). However, the in situ
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8.
PENTCHEV ET AL. Table I.
Lysosomal
Blockage
Demonstrated Lysosomal Uptake of Exogenously Administered Enzymes
Enzyme
Reference
Tissue
3-Glucuronidase
Cultured
a-Glucosidase
Rat Liver
(4)
Glucocerebrosidase
Rhesus Monkey Liver
(5)
Hexosaminidase
Rat Liver
(6)
Table II.
Fibroblasts
(3)
Experimental Models for Correction of Lysosomal Accumulation with Exogenous Enzymes
Effect
Reference No.
Invertase and 3-glucosidase
Reversal of Sucrose and cellobiose accumulation in macrophases
7
Deficient lysosomal hydrolases:
Metabolic correction in patients' fibroblasts:
Mucopolysacchari dases
Mucopolysacchari doses
8
Arylsulfatase-A
Metachromatic leukodystrophy
9
Acid lipase
Wolman's Disease
10
ofr-Galactosidase
Fabry's Disease
11
3-N-Acetylhexosaminidase
Sandhoff Disease
Enzyme Addition
B. Invertase
Dextranase
12
Animals
Prevention of hepatic sucrose accumulation in rat
13
Reversal of hepatic dextran accumulation in rat
14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
154
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Table III.
Initial Results of Enzyme Replacement Trials in Gaucher's Disease (17)
1.
Intravenous infusions of highly purified human placental glucocerebrosidase were well tolerated by patients with no untoward immunological or pyrogenic response.
2.
Exogenous enzyme tion.
3.
A clearance of the elevated glycolipid levels was observed in the blood stream.
4.
Following enzyme infusion, lower levels of glycolipid were found in l i v e r biopsy samples.
Table IV.
Assessment of Lysosomal Responses in Evaluating the Effectiveness of Enzyme Replacement for the Treatment of Storage Disorders
1.
The extent of enzymatically induced clearance of stored material.
2.
The rate at which stored material
3.
The degree of lysosomal overloading in relationship to c l i n i c a l and pathological manifestations.
4.
The c l i n i c a l consequence of reversing intralysosomal accumulation.
accumulates.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PENTCHEV ET AL.
Lysosomal
Blockage
Figure 2. Clearance of glucocerebroside from Gaucher liver tissue by exogenous glucocerebrosidase in vitro. Fresh liver needle biopsy samples from a Gaucher patient were incubated in phosphate-buffered saline at pH = 6.0 for 12 hr at 37°C without and with 1000 units of highly purified glucocerebrosidase as indicated. Glucocerebroside in these preparations and in nonincubated samples was subsequently quantitated as previously described (23). Gl I = glucocerebroside; Cer-ceramide, PE = phosphatidyl ethanolamine.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Adult
Adult
Adult
F
F
M
M
F
F
F
F
F
3
4
5
6
7
8
9
10
Π
F&M
Adult
M
2
itrol
Adult
F
1
Adult
Adult
Adult
Adult
Juvenile
Adult
Sex
Patient
Type
Table V.
20-35
21
23
49
39
30
20
30
15
54
15
44
Age
100
29
25
23
23
23
0.03 - 0.07
22.90
17.90
17.80
16.50
16.40
15.40
11.20
-27
4.30
1.63
0.70
0.25
(mg/gram wet t i s s u e )
Liver Glucocerebroside
29
30
21
16
(% of Control)
Residual Glucocerebrosidase A c t i v i t y i n Leukocytes
Glycolipid Accumulation in Livers of Gaucher Patients
8.
PENTCHEV ET AL.
Lysosomal
Blockage
157
accessability of l i p i d may be more restrictive and perhaps a limiting factor. Preliminary studies with mouse l i v e r have shown that residual bodies have a lower tendency to fuse with lysosomes containing exogenous material (24). Secondly, the efficiency and course of enzyme replacement regimens w i l l depend on the degree to which an induced clearance can be sustained. Obviously, a rapid rate of accumulation relative to clearance w i l l limit the u t i l i t y of this form of treatment. It may be useful to attempt to stabilize the exogenous enzyme in order to prolong its intralysosomal digestive effect. The h a l f - l i f e of human glucocerebrosidase in the l i v e r of rats and Rhesus monkeys is approximately 8 hours (5). Generalized hypothermia (33°C) extends this halfl i f e to over thirty hours (5). Lipids have also been shown to stabilize the enzyme in vitro (Furbish, F. and Brady, R., unpublished observations). Stabilization and protection of the enzyme may also be achieved by cross-linking or binding to matrices (25). Thirdly, the progressio c l i n i c a l disease states remains obscure and speculative. The v a r i a b i l i t y of biochemical and c l i n i c a l manifestations in Gaucher's is i l l u s t r a t i v e of this gap in our knowledge. The range of elevated glucocerebroside in the l i v e r of patients with Gaucher's disease may vary over 100-fold (Table V). Often l i t t l e correlation was observed between the degree of glucocerebroside accumulation and the severity of c l i n i c a l manifestations. Accordingly, a given level of clearance induced by enzyme supplementation may show considerable variation in terms of c l i n i c a l benefits. F i n a l l y , i t has sometimes been t a c i t l y assumed that an enzymatically induced reversal of lysosomal blockage w i l l restore normality to the effected tissues. As already discussed, the reality of biochemically defined corrections has been well established with various models. However, descriptions of morphological correlates to biochemical correction are rather sparce in the literature. The classic experiments of Cohn et a l . {7) reporting a visual observation of the reversal of lysosomal swelling in the sucrose-loaded macrophages upon the uptake by the cells of invertase remains to date the most descriptive accounting of this phenomena. The complexities of biochemical, morphological and physiological relationships in organisms are enormous. The present status of our knowledge is not sufficient to evaluate potentially corrective or simply preventive roles for enzyme replacement. In conclusion, the r e a l i s t i c application of enzyme replacement as a therapeutic measure for the treatment of lysosomal storage disorders has yet to be established. Although the present state of the art is at a delicate embryonic stage of development, nature has extended a helpful hand in providing intracellular homing mechanisms. The continued creative and persistent efforts of investigators can l i k e l y complement this advantage towards a meaningful realization of this form of therapy.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
158
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
REFERENCES 1.
Brady, R., Metabolism 26: 329 (1977).
2. 3.
DeDuve,C.;Fed. Proc. 23, 1045 (1964). Lagunoff, D., Nicol, D., Prilzl, P., Lab. Invest. 29: 449 (1973).
4.
Huijing, F., Waltuck, B., Whelan, W., in Birth Defects Origi nal Article Series, Enzyme Therapy in Genetic Diseases, Vol. IX, No. 2, p. 191, (1973). Daniel Bergsma, Ed.
5.
Pentchev, P., Kusiak, J., Barranger, J., Furbish, F., Rapoport,S., Massey, J., Brady, R., in Enzymes of Lipid Metabolism, Plenum Press i Press 6. Schlesinger, P., Rodman, J., Frey, M., Lang, S., Stahl, P., Arch. Biochem. and Biophys. 172: 606 (1976). 7.
Cohn, Z., Ehrenreich, B., J. Exp. Med. 129: 201 (1969).
8.
Neufeld, L., Shapiro, L., Ann. Rev. Biochem. 44: 357 (1975).
9.
Porter, M., Fluharty, Α., Kihara, H., Science 172: 1263 (1971).
10. Kyriakides, E., Paul, B., Balint, J., J. Lab. Clin. Med. 80: 810 (1972). 11. Dawson, G., Matalon, R., Li, Y., Pediat. Res. 7: 684 (1973). 12. Cantz, M., Kresse, H., J. Biochem. 47: 581 (1974). 13. Jacques, P., Bruns, G., Abs. 2nd Meet. Fed. Eur. Biochem. Soc. A36 (1978) 14. Colley, M., Ryman, Β., Bioch. and Biophys. Acta. 451: 417 (1976). 15. Brady, R., N. Engl. J. Med. 275: 312 (1966). 16. Pentchev, P., Brady, R., Hibbert, S., Gal, Α., Shapiro, D., J. Biol. Chem. 248: 5256 (1973). 17. Brady, R., Pentchev, P., Gal, Α., Fed. Proc. 34: 1310 (1975). 18. Furbish, F., Blair, H., Shiloach, J., Pentchev, P., Brady, R. Proc. Natl. Acad. Sci. USA 74: 3560 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8. PENTCHEV ET AL.
Lysosomal Blockage
159
19. Belchetz, P., Crawley, J., Braidman, I., Gregoriadis, G. Lancet ii, 116 (1977). 20. Beutler, E., Dale, G., Guinto, E., Kuhl, W. Proc. Natl. Acad. Sci. USA, 74: 4620 (1977). 21. Furbish, F., Steer, C., Barranger, J., Jones, J., Brady, R. Biochem. Biophys. Res. manuscript submitted. 22. Brady, R., King, F. In Lysosomes and Storage Diseases (1973) H. G. Hers and F. Van Hoof, Editors, Academic Press, New York, p. 381. 23. Brady, R., Pentchev, P., Gal, Α., Hibbert, S., Dekaban, Α., Ν. Engl. J. Med. 291: 989 (1974). 24. Daems, W., in Enzym p. 3, (1974), J. M. Tager, G. S. Hooghwinkel and W. Daems, Editors, North-Holland Publishing Company. 25. Wold, F., in Birth Defects, Original Article Series, Vol. IX, No. 2, Enzyme Therapy in Genetic Diseases, p. 46. (1973). Daniel Bergama, Editor, Williams and Wi1 king Publishers, Baltimore, Md. RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9 Blood Coagulation—Initiation and Regulation by Limited Proteolysis WALTER KISIEL Department of Biochemistry, University of Washington, Seattle, WA 98195
During the coagulation of blood, a number of plasma glycoprotein zymogens are converted to serine proteases in the presence of calcium ions and phospholipid. These activation reactions do not occur randomly but rather as a series of consecutive zymogen activation reactions where the product of one reaction is the catalyst for the next. The focal point of this cascade-like reaction is the formation of thrombin, the serine protease which converts fibrinogen to fibrin. In vivo, the formation of fibrin is thought to occur on the surface of platelets concurrent with their response to an injury in the vessel wall. Indeed, platelets constitute the primary defense in arresting the bleeding of ruptured blood vessels by their rapid adhesion to extravascular surfaces. The platelet adhesion reaction is followed by a morphological change in the platelet membrane which provides a catalytic phospholipid surface for optimal generation of thrombin. The ensuing fibrin serves to stabilize the hemostatic platelet thrombus by virtue of its mesh-like properties. Following tissue repair and vessel wall regeneration, the hemostatic plug is destabilized by the degradation of the fibrin network by plasmin arising from the fibrinolytic system. The common names, as well as the Roman numeral designations, of the glycoproteins participating in coagulation are listed in Table I. Prothrombin, factor VII, factor IX, factor X, factor XI, factor XII, and prekallikrein exist in plasma as precursors to serine proteases. Also listed in Table I are the coagulation disorders associated with the deficiency of a particular coagulation factor. In the past decade, enormous progress has been made in the isolation and characterization of most of these proteins. The availability of homogeneous preparations of each zymogen has made is possible to establish unambiguously the mechanism whereby it is activated, and its proteolytic specificity following activation. The results of these studies have proven invaluable in our understanding of the molecular events in coagulation, and 0-8412-0452-7/78/47-080-160$05.00/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
161
Coagulation
TABLE I The
P r o t e i n s o f Blood
Coagulation
Roman Numeral Des i g n a t i o n
Associated
Common Name
Disorder
Fibrinogen
Factor 1
Afibrinogenemia
Prothrombi n
F a c t o r 11
Prothrombin d e f i c i e n c y
Proaccelerin
Factor V
Parahemophi1ia
Proconvertin
F a c t o r VI1
ProSPCA d e f i c i e n c y
Factor
Hemophi1ia A
Tissue
Factor
Antihemophi1ic Christmas
Factor
F a c t o r IX
Christmas disease ( h e m o p h i l i a B)
Factor
Factor X
Stuart factor deficiem
Plasma T h r o m b o p l a s t i n A n t e c e d e n t (PTA)
F a c t o r XI
PTA
Hageman
F a c t o r XI1
Hageman
Factor
Factor XIII d e f i c i e n c y
Stuart
Factor
VIII
Factor
Fibrin-stabi1izing Factor Prekal1ikrein Factor)
XIII
deficiency trait
(Fletcher
HMW K i n i n o g e n (high molecular weight k i n i nogen, Fitzgerald factor)
Fletcher factor Fitzgerald
disease
trait
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
162
have g r e a t l y a s s i s t e d t h e r e s e a r c h e r i n f o r m u l a t i n g a more I n t e l l i g e n t approach t o the understanding o f the k i n e t i c s o f the overa l l c o a g u l a t i o n p r o c e s s in vivo. F u r t h e r m o r e , f r o m such s t u d i e s has emerged t h e c o n c e p t t h a t t h e p r i n c i p a l mechanism f o r t h e i n i t i a t i o n , p r o p a g a t i o n and r e g u l a t i o n o f t h e c o a g u l a t i o n process i n v o l v e s t h e s e l e c t i v e e n z y m a t i c c l e a v a g e o f u n i q u e p e p t i d e bonds. In t h i s a r t i c l e , t h e p r i m a r y e m p h a s i s w i l l be p l a c e d on more r e c e n t i n f o r m a t i o n r e g a r d i n g t h e mechanism o f a c t i v a t i o n o f e a c h c o a g u l a t i o n zymogen l e a d i n g t o f i b r i n f o r m a t i o n . In a d d i t i o n , c e r t a i n a c t i v a t e d c o a g u l a t i o n f a c t o r s have been o b s e r v e d t o c a r r y o u t s e c o n d a r y p r o t e o l y t i c r e a c t i o n s i n t h e t e s t t u b e and t h e s e w i l l be p r e s e n t e d and d i s c u s s e d i n r e l a t i o n t o t h e i r p o t e n t i a l r e g u l a t o r y r e l e v a n c e in vivo. I I.
M o l e c u l a r Events i n Blood
Coagulation
Two pathways a p p a r e n t l the s e r i n e p r o t e a s e w h i c pathways have been r e f e r r e d t o a s t h e i n t r i n s i c and e x t r i n s i c c o a g u l a t i o n p a t h w a y s . The i n t r i n s i c pathway i n c l u d e s t h o s e r e a c t i o n s t h a t l e a d t o f a c t o r Xa f o r m a t i o n u t i l i z i n g f a c t o r s p r e s e n t o n l y i n p l a s m a . These f a c t o r s i n c l u d e f a c t o r X I I , f a c t o r X I , f a c t o r IX and f a c t o r V I M . P r e s e n t l y , i t i s not c l e a r whether h i g h m o l e c u l a r w e i g h t k i n i n o g e n and p r e k a l 1 i k r e i n a r e a l s o r e q u i r e d f o r t h i s pathway inasmuch a s p l a s m a l a c k i n g t h e s e p r o t e i n s c l o t s p o o r l y upon e x p e r i m e n t a l l y i n i t i a t i n g t h e i n t r i n s i c s y s t e m . The e x t r i n s i c pathway o f f a c t o r Xa g e n e r a t i o n i n v o l v e s t h e p a r t i c i p a t i o n o f p l a s m a f a c t o r V I I and t i s s u e f a c t o r , a s p e c i f i c l i p o p r o t e i n r e l e a s e d f r o m t i s s u e s u r r o u n d i n g t h e damaged b l o o d vessel w a l l . These pathways c l e a r l y c a n be d i f f e r e n t i a t e d i n t h e t e s t t u b e d e p e n d i n g on t h e i n i t i a t i n g s t i m u l u s p r o v i d e d . Factor Xa formed by e i t h e r t h e i n t r i n s i c o r e x t r i n s i c pathway a p p e a r s biochemically indistinguishable. Following i t s formation, factor Xa t h e n t r i g g e r s a s e q u e n c e o f r e a c t i o n s w h i c h r e s u l t s i n t h e formation of insoluble f i b r i n . I t i s perhaps noteworthy t o m e n t i o n t h a t e a c h o f t h e s e pathways I s t h o u g h t t o be t r i g g e r e d by t h e i r i n i t i a l i n t e r a c t i o n w i t h e l e m e n t s o f t h e v a s c u l a r subendothelium. The r a t e and i n t e n s i t y o f f a c t o r Xa f o r m a t i o n in vivo c o n c e i v a b l y i s d i c t a t e d by t h e pathway f o l l o w e d w h i c h i n t u r n i s r e l a t e d t o t h e s e v e r i t y o f t h e v a s c u l a r trauma. Thus, v a s c u l a r trauma w h i c h i n v o l v e s a b r e a k i n t h e c o n t i n u i t y o f t h e v e s s e l with concomitant release o f t i s s u e f a c t o r necessitates immediate f o r m a t i o n o f f a c t o r Xa w h i c h p r e s u m a b l y o c c u r s v i a e x t r i n s i c clotting. On t h e o t h e r h a n d , r e l a t i v e l y m i l d v a s c u l a r trauma i n w h i c h t h e v e s s e l does n o t l o s e i t s c o n t i n u i t y , may c a l l f o r a r e l a t i v e l y s l o w , more c o n t r o l l e d f o r m a t i o n o f f a c t o r Xa. Under t h e s e c i r c u m s t a n c e s , f a c t o r Xa c o u l d be formed v i a t h e i n t r i n s i c pathway i n i t i a t e d by t h e i n t e r a c t i o n o f f a c t o r X I I w i t h a s u b endothelial collagen surface.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
A.
KISIEL
Blood
163
Coagulation
The A c t i v a t i o n o f F a c t o r X by t h e
I n t r i n s i c Pathway
The s e q u e n c e o f e v e n t s w h i c h l e a d t o t h e f o r m a t i o n o f f a c t o r Xa by t h e i n t r i n s i c pathway o f b l o o d c o a g u l a t i o n is schematically r e p r e s e n t e d i n F i g u r e 1, The f i r s t s t e p i n t h e s e c a s c a d i n g r e a c t i o n s i s the a c t i v a t i o n o f f a c t o r X I I . F a c t o r X I I has been i s o l a t e d f r o m human and b o v i n e p l a s m a and c h a r a c t e r i z e d i n r e g a r d t o i t s s i z e , s t r u c t u r e and c o m p o s i t i o n (J_,.2). F a c t o r XII i s o l a t e d f r o m e i t h e r s o u r c e a p p e a r s t o be a s i n g l e - c h a i n glycop r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 75,000. F a c t o r XI l a h a s a l s o been i s o l a t e d f r o m b o v i n e p l a s m a by F u j i k a w a e t a l . ( 3 ) . B o v i n e f a c t o r XI l a i s composed o f a heavy and a l i g h t c h a i n h e l d t o g e t h e r by d i s u l f i d e bonds. The l i g h t c h a i n h a s a m o l e c u l a r w e i g h t o f 28,000 and c o n t a i n s an a m i n o - t e r m i n a l s e q u e n c e o f Val-Val-Gly-Gly ( 3 ) , The a m i n o - t e r m i n a l s e q u e n c e o f the heavy c h a i n (mol wt 46,000) i s T h r - P r o - P r o - T r p - L y s - and i s i d e n t i c a l t o t h a t o b s e r v e d f o r the s i n g l e - c h a i n The c a r b o x y l - t e r m i n a l residu T h u s , the c o n v e r s i o n o f b o v i n e f a c t o r X I I t o f a c t o r XI l a i s due t o t h e c l e a v a g e o f a s i n g l e i n t e r n a l a r g i n y l - v a l i n e p e p t i d e bond. F a c t o r XI l a e x h i b i t e d a m i d a s e a c t i v i t y t o w a r d arginine-containing s u b s t r a t e s and i s r e a d i l y i n h i b i t e d by t h e s e r i n e p r o t e a s e i n h i b i t o r , d i i s o p r o p y l p h o s p h o f l u o r i d a t e (DFP). T h i s i n h i b i t o r i s bound e x c l u s i v e l y t o t h e l i g h t c h a i n o f f a c t o r XI l a i n d i c a t i n g t h a t the a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h i s c h a i n . Furthermore, the a c t i v e s i t e region i n t h e l i g h t c h a i n o f f a c t o r XI l a c o n t a i n s an A s p - A l a - C y s - G l n - G l y - A s p - S e r - G l y - G l y - s e q u e n c e w h i c h i s t y p i c a l o f the o t h e r p l a s m a s e r i n e p r o t e a s e s ( 2 ) . The mechanism whereby f a c t o r X I I i s a c t i v a t e d p h y s i o l o g i c a l l y i s p r e s e n t l y n o t c l e a r . M o r e o v e r , the p r o t e a s e ( s ) w h i c h c a r r i e s o u t t h i s r e a c t i o n has n o t been i d e n t i f i e d . The a c t i v a t i o n o f f a c t o r X I I h a s been t h o u g h t t o o c c u r in vivo f o l l o w i n g i t s i n t e r a c t i o n with a charged s u r f a c e which presumably i s s u b e n d o t h e l i a l collagen. The a c t i v a t i o n o f f a c t o r X I I has been i n v e s t i g a t e d i n the t e s t tube u t i l i z i n g g l a s s o r k a o l i n as the n e g a t i v e l y charged surface. I n c u b a t i o n o f s i n g l e - c h a i n f a c t o r XII w i t h k a o l i n prod u c e s a [ f a c t o r X l l - k a o l i n ] c o m p l e x w h i c h does n o t have e n z y m a t i c a c t i v i t y towards small s u b s t r a t e s . In a d d i t i o n , t h i s c o m p l e x i s not i n h i b i t e d by DFP s u g g e s t i n g t h a t f a c t o r X I I i s n o t c o n v e r t e d t o an a c t i v e enzyme s i m p l y by i t s i n t e r a c t i o n w i t h t h e k a o l i n surface. However, t h e [ f a c t o r X l l - k a o l i n ] c o m p l e x w i l l react w i t h f a c t o r XI and p r e k a l 1 i k r e i n c o n v e r t i n g t h e s e zymogens t o a c t i v e enzymes ( 3 ) . Whether f a c t o r X I I was c o n v e r t e d t o f a c t o r XI l a d u r i n g t h i s i n c u b a t i o n i s n o t known. G r i f f i n and C o c h r a n e (4) and Chan et^ aj_. (5) have r e p o r t e d t h a t human f a c t o r X I I i s a c t i v a t e d i n t h e p r e s e n c e o f k a i l i k r e i n , HMW k i n i n o g e n and k a o l i n . These s t u d i e s s u g g e s t t h a t f a c t o r X I I i n t e r a c t s s t o i c h i o m e t r i c a l l y w i t h HMW k i n i n o g e n on t h e k a o l i n s u r f a c e and i s s u b s e q u e n t l y a c t i v a t e d by k a l l i k r e i n . From t h i s i n f o r m a t i o n , i t i s c o n c e i v a b l e t h a t upon i n t e r a c t i o n w i t h a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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c h a r g e d s u r f a c e i n the p r e s e n c e o f HMW k i n i n o g e n , f a c t o r X\\ u n d e r g o e s a c o n f o r m a t i o n a l change w h i c h p e r m i t s i t s a c t i v e s i t e t o r e a c t s l o w l y and p r e f e r e n t i a l l y w i t h p r e k a l 1 i k r e i n . The r e l a t i v e l y s m a l l amount o f k a l l i k r e i n formed can r e c i p r o c a l l y a c t i v a t e s u b s t a n t i a l amounts o f f a c t o r X I f . Once formed i n s u f f i c i e n t l e v e l s , f a c t o r XI l a c a n * t h e n c a t a l y z e t h e a c t i v a t i o n o f t h e n e x t zymogen i n t h e c a s c a d e , namely f a c t o r X I . F a c t o r XI i s c o n v e r t e d t o f a c t o r X l a by f a c t o r XI l a , a r e a c t i o n a p p a r e n t l y augmented by HMW k i n i n o g e n and k a o l i n ( 6 ) . F a c t o r XI l a i s p r e s e n t l y t h e o n l y c o a g u l a t i o n p r o t e a s e known w h i c h can a c t i v a t e f a c t o r XI i n t h e t e s t t u b e . The a p p a r e n t o b l i g a t o r y r o l e o f f a c t o r XI l a i n the a c t i v a t i o n o f f a c t o r XI i s i n t e r e s t i n g i n v i e w o f the f a c t t h a t p a t i e n t s w i t h f a c t o r X I I d e f i c i e n c y have no d e m o n s t r a t i v e b l e e d i n g t e n d e n c i e s , y e t p a t i e n t s w i t h f a c t o r XI d e f i c i e n c y o f t e n b l e e d c o n s i d e r a b l y f o l l o w i n g i n j u r y or minor s u r g e r y . On possibl explanatio fo thi phe nomenon i s t h a t f a c t o r X I l - d e f i c i e n l e v e l s o f f a c t o r XII t coagulation g coagu l a t i o n p r o c e s s i s p r o p a g a t e d by an a m p l i f i c a t i o n mechanism, the s m a l l e s t l e v e l o f enzyme r e q u i r e d w o u l d be i n t h e i n i t i a l r e actions. An a l t e r n a t i v e e x p l a n a t i o n i s t h a t o t h e r h e r e t o f o r e unr e c o g n i z e d f a c t o r s may p l a y a m a j o r r o l e i n the p h y s i o l o g i c a l a c t i v a t i o n of f a c t o r XI, In t h i s c a s e , p r o t e o l y t i c c l e a v a g e o f f a c t o r XI may be a s e c o n d a r y r e a c t i o n c a t a l y z e d by f a c t o r XI l a and t h a t o t h e r p r o t e i n s s u c h as p r e k a 1 1 i k r e i n and p l a s m i n o g e n p r o a c t i v a t o r c o n s t i t u t e more s p e c i f i c p r i m a r y s u b s t r a t e s for f a c t o r XI l a . F a c t o r XI has been i s o l a t e d f r o m human and b o v i n e p l a s m a . Human f a c t o r XI i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 124,000. I t i s composed o f two i d e n t i c a l p o l y p e p t i d e c h a i n s (mol wt a b o u t 60,000) h e l d t o g e t h e r by d i s u l f i d e bonds. D u r i n g t h e a c t i v a t i o n o f f a c t o r XI an i n t e r n a l p e p t i d e bond i n e a c h o f the two c h a i n s i s c l e a v e d by f a c t o r XI l a ( 7 ) . T h i s g i v e s r i s e t o two heavy c h a i n s (mol wt 35,000) and two l i g h t c h a i n s (mol wt 25,000) in f a c t o r X l a . The a m i n o - t e r m i n a l amino a c i d s e q u e n c e o f the l i g h t chains in f a c t o r Xla i s I l e - V a l - G l y - G l y . T h i s sequence shows homology w i t h o t h e r p l a s m a s e r i n e p r o t e a s e s i n v o l v e d i n coagulation. The l i g h t c h a i n s a l s o c o n t a i n the a c t i v e s i t e s e r i n e w h i c h r e a c t s q u a n t i t a t i v e l y w i t h DFP. Thus, f a c t o r X l a i s a s e r i n e p r o t e a s e c o n t a i n i n g two a c t i v e s i t e s per m o l e c u l e o f enzyme, and r e p r e s e n t s t h e f i r s t c a s e o f a s e r i n e p r o t e a s e w i t h two c a t a l y t i c s i t e s ( 7 ) . The n e x t zymogen t o be a c t i v a t e d i n t h e i n t r i n s i c pathway i s f a c t o r IX. Human and b o v i n e f a c t o r IX have been i s o l a t e d and w e l l c h a r a c t e r i z e d i n t h e p a s t few y e a r s (8^,9). B o v i n e f a c t o r IX i s a s i n g l e - c h a i n g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000. Human f a c t o r IX i s a l s o a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 57,000 and p o s s e s s e s an a m i n o - t e r m i n a l amino a c i d s e q u e n c e n e a r l y i d e n t i c a l t o t h a t o f b o v i n e f a c t o r IX ( 9 ) .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
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F a c t o r IX i s one o f t h e f o u r c o a g u l a t i o n f a c t o r s w h i c h r e q u i r e v i t a m i n K f o r i t s b i o s y n t h e s i s . The o t h e r f a c t o r s i n c l u d e p r o t h r o m b i n , f a c t o r X and f a c t o r Vitamin K p a r t i c i p a t e s i n the b i o s y n t h e s i s o f these p r o t e i n s i n t h e post-ribosoma1 carboxyl a t i o n o f s p e c i f i c g l u t a m i c a c i d r e s i d u e s p r e s e n t i n t h e aminoterminal p o r t i o n o f these p r o t e i n s (10). The new amino a c i d , y-carboxyglutamic a c i d , i s e s s e n t i a l f o r c a l c i u m i o n b i n d i n g by t h e s e p r o t e i n s and t h e i r s u b s e q u e n t I n t e r a c t i o n w i t h a c i d i c p h o s p h o l i p i d membranes. The y - c a r b o x y g l u t a m i c a c i d residues i n these p r o t e i n s tend t o o c c u r as a d j a c e n t r e s i d u e s w h i c h f a c i l i t a t e t h e c o o r d i n a t i o n o f one c a l c i u m i o n p e r p a i r o f y - c a r b o x y g l u t a m i c a c i d r e s i d u e s (JJ_). F a c t o r IX i s a c t i v a t e d by f a c t o r X l a i n t h e p r e s e n c e o f c a l cium ions i n a two-step process. In t h e f i r s t s t e p , s i n g l e - c h a i n f a c t o r IX i s c o n v e r t e d t o a t w o - c h a i n m o l e c u l e by t h e c l e a v a g e o f an i n t e r n a l A r g - A l a p e p t i d a c t i o n has no e n z y m a t i (mol wt 16,000) w i t h an a m i n o - t e r m i n a l sequence o f T y r - A s n - S e r G l y , and a heavy c h a i n (mol wt 38,000) w i t h an a m i n o - t e r m i n a l sequence o f A l a - G l y - T h r - I l e . T h i s i n t e r m e d i a t e i s then c o n v e r t e d t o f a c t o r IXa i n a r a t e - l i m i t i n g s t e p by l i m i t e d p r o t e o l y s i s . In t h i s r e a c t i o n , a s p e c i f i c A r g - V a l bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e h e a v y c h a i n i s c l e a v e d by f a c t o r X l a w i t h t h e c o n c o m i t a n t r e l e a s e o f an a c t i v a t i o n p e p t i d e . T h u s , t h e f a c t o r IXa formed i n t h i s r e a c t i o n i s composed o f two c h a i n s h e l d t o g e t h e r by d i s u l f i d e bonds. The heavy c h a i n (mol wt 27,300) c o n t a i n s t h e active s i t e serine residue and c o n t a i n s an a m i n o - t e r m i n a l s e quence o f V a l - V a l - G l y - G l y . The l i g h t c h a i n (mol wt 1 6 , 6 0 0 ) , w h i c h o r i g i n a t e s f r o m t h e a m i n o - t e r m i n u s o f t h e zymogen, c o n t a i n s the y-carboxyglutamic a c i d r e s i d u e s and t h e a m i n o - t e r m i n a l s e quence o f T y r - A s n - S e r - G l y ( l _ 2 ) . F a c t o r IXa i s t h e s e r i n e p r o t e a s e w h i c h c o n v e r t s f a c t o r X t o f a c t o r Xa by l i m i t e d p r o t e o l y s i s i n t h e i n t r i n s i c pathway. In t h i s r e a c t i o n , f a c t o r IXa f o r m s a m a c r o m o l e c u l a r c o m p l e x w i t h an a d d i t i o n a l p r o t e i n , f a c t o r V I I I , i n the presence o f p h o s p h o l i p i d and c a l c i u m i o n s . F a c t o r V I I I i s t h e plasma p r o t e i n t h a t i s a b s e n t i n i n d i v i d u a l s w i t h c l a s s i c h e m o p h i l i a , t h e most p r e v a l e n t c o a g u l a t i o n d i s o r d e r known. F a c t o r V I I I has been i s o l a t e d f r o m human and b o v i n e plasma and e x h i b i t s a m o l e c u l a r w e i g h t o f a b o u t one m i l l i o n (l_3,]_4). These p r e p a r a t i o n s p o s s e s s potent coagulant a c t i v i t y a s w e l l as von W i l l e b r a n d p l a t e l e t a g g r e g a t i n g a c t i v i t y . Treatment o f t h e bovine f a c t o r V I I I p r e p a r a t i o n w i t h c a t a l y t i c amounts o f b o v i n e t h r o m b i n r e s u l t s i n a 2 5 - f o l d i n c r e a s e i n f a c t o r V I I I procoagulant a c t i v i t y i n t h e presence o f CaCl2. Gel f i l t r a t i o n o f t h i s thrombin-treated preparation o f f a c t o r V I M r e s u l t s i n t h e s e p a r a t i o n o f f a c t o r V I M c o a g u l a n t a c t i v i t y and the p l a t e l e t a g g r e g a t i n g a c t i v i t y (l_5). However, no d e t e c t a b l e p r o t e i n was a s s o c i a t e d w i t h t h e f a c t o r V I M c o a g u l a n t a c t i v i t y . T h i s has l e d t o s p e c u l a t i o n t h a t t h e f a c t o r V I M r e p r e s e n t s o n l y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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a small f r a c t i o n o f the t o t a l p r o t e i n i n the f a c t o r V M t prepar a t i o n s d e s c r i b e d (15). The r o l e o f f a c t o r V I M in the a c t i v a t i o n o f f a c t o r X appears t o be one o f a r e g u l a t o r y n a t u r e . Conceivably, factor VMI could augment t h i s r e a c t i o n by p r o m o t i n g t h e f o r m a t i o n o f a more r e a c t i v e c o n f o r m a t i o n i n e i t h e r f a c t o r IXa o r i t s s u b s t r a t e , f a c t o r X. The f i n a l s t e p i n t h i s pathway i s t h e a c t i v a t i o n o f f a c t o r X, F a c t o r X i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000 and one o f t h e most w e l l c h a r a c t e r i z e d p r o t e t n s i n b l o o d coagulation. I n d e e d , t h e t o t a l amino a c i d s e q u e n c e o f b o v i n e f a c t o r X has been completed (16). i t c o n s i s t s o f a h e a v y c h a i n (mol wt 39,300) and a l i g h t c h a i n (mol wt 16,500) h e l d t o g e t h e r by d i s u l f i d e b o n d s . The h e a v y c h a i n c o n t a i n s 307 amino a c i d r e s i d u e s w i t h two g l y c o s y l m o i e t i e s l i n k e d t o Asn-35 and T h r - 3 0 0 ( 1 6 ) . The heavy c h a i n a l s o c o n t a i n s the a c t i v e s i t serin located I p o s i t i o 233 Th l i g h t c h a i n c o n t a i n s th acid residues. The c o n v e r s i o n o f b o v i n e f a c t o r X t o f a c t o r Xa by f a c t o r IXa o c c u r s as a r e s u l t o f t h e c l e a v a g e o f a s i n g l e p e p t i d e bond between Arg-51 and M e - 5 2 , T h i s r e s u l t s i n t h e r e l e a s e o f an a c t i v a t i o n p e p t i d e (mol wt 10,500) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f the heavy c h a i n a n a l o g o u s t o t h a t o b s e r v e d in the a c t i v a t i o n o f f a c t o r IX. F a c t o r Xa i s a s e r i n e p r o t e a s e w h i c h , i n t h e p r e s e n c e o f f a c t o r V, p h o s p h o l i p i d and c a l c i u m i o n s , c a t a l y z e s t h e c o n version of prothrombin to thrombin. B.
The
Activation
of
F a c t o r X by
the
Extrinsic
Pathway
The p r o p o s e d s e r i e s o f r e a c t i o n s w h i c h r e s u l t i n t h e a c t i v a t i o n o f f a c t o r X by t h e e x t r i n s i c pathway a r e d e p i c t e d i n F i g u r e 2. In t h e s e r e a c t i o n s , p l a s m a f a c t o r V I I , i n t h e p r e s e n c e o f c a l c i u m i o n s , f o r m s a c o m p l e x w i t h a l i p o p r o t e i n r e l e a s e d f r o m damaged tissue. T h i s c o m p l e x s u b s e q u e n t l y c o n v e r t s f a c t o r X t o f a c t o r Xa which appears i n d i s t i n g u i s h a b l e from t h a t observed f o l l o w i n g i n t r i n s i c a c t i v a t i o n o f f a c t o r X (T7). Present evidence suggests that f a c t o r VII i s c o n v e r t e d to f a c t o r V i l a in o r d e r to c a t a l y z e this reaction. The l i p o p r o t e i n s e r v e s as a h i g h m o l e c u l a r w e i g h t c o f a c t o r which i s a b s o l u t e l y e s s e n t i a l f o r the e x p r e s s i o n o f factor Vila proteolytic activity. F a c t o r VII i s a t r a c e plasma g l y c o p r o t e i n . I t s low c o n c e n t r a t i o n has v i r t u a l l y p r e c l u d e d t h e i s o l a t i o n o f t h i s p r o t e i n f r o m human p l a s m a . F a c t o r V I I has been i s o l a t e d f r o m b o v i n e plasma as a s i n g l e - c h a i n p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 45,500 (l_8,l_9). In the t e s t t u b e , s i n g l e - c h a i n f a c t o r V I I can be c o n v e r t e d t o a t w o - c h a i n p r o t e i n w h i c h e x h i b i t s 30-40 t i m e s t h e c o a g u l a n t a c t i v i t y o f t h e s i n g l e - c h a i n f o r m . The a c t i v a t i o n o f f a c t o r V I I , t h a t i s , t h e f o r m a t i o n o f t w o - c h a i n f a c t o r V I I , has been o b s e r v e d f o l l o w i n g t h e i n c u b a t i o n o f f a c t o r V I I w i t h f a c t o r XI l a ( 2 0 ) , f a c t o r X l a ( 2 0 , k a l l i k r e i n ( 2 J J , f a c t o r Xa ( 2 2 ) , and
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
167
Coagulation
t h r o m b i n (22). F a c t o r V M a i s composed o f a h e a v y c h g i n (mol wt 34,000) and a l i g h t c h a i n (mol wt 23,000) h e l d t o g e t h e r by d i s u l f i d e bonds. The a c t i v e s i t e s e r i n e i s l o c a t e d In t h e heavy c h a i n which c o n t a i n s t h e a m i n o - t e r m i n a l sequence o f I l e - V a l - G l y Gly (20). The l i g h t c h a i n c o n t a i n s t h e y - c a r b o x y g l u t a m i c a c i d r e s i d u e s and a r i s e s f r o m t h e a m i n o - t e r m i n a l p o r t i o n o f t h e s i n g l e c h a i n zymogen ( 2 0 ) , The c a r b o x y l - t e r m i n a l residue o f the l i g h t chain i s arginlne. Thus, f a c t o r V t l a Is a s e r i n e p r o t e a s e a r i s ing from t h e p r o t e o l y s i s o f a s i n g l e i n t e r n a l A r g - l l e p e p t i d e bond. F a c t o r V i l a has an a b s o l u t e r e q u i r e m e n t f o r t i s s u e f a c t o r i n o r d e r t o c l e a v e t h e A r g - 5 1 - M e - 5 2 p e p t i d e bond In t h e heavy c h a i n o f f a c t o r X, The p h y s i o l o g i c a l mechanism f o r t h e a c t i v a t i o n o f f a c t o r V I I i s p r e s e n t l y n o t known. W h i l e s e v e r a l s e r i n e p r o t e a s e s t h a t p a r t i c i p a t e i n t h e I n t r i n s i c pathway w i l l c o n v e r t f a c t o r V M t o f a c t o r V i l a in vitvo i t i c i p a t e i n theactivatio factor X i stheonly protei t h e p r o t e o l y t i c r a n g e o f f a c t o r V M a has n o t been Investigated, W h i l e c o n s i d e r a b l e i n f o r m a t i o n h a s a c c u m u l a t e d on t h e m o l e c u l a r p r o p e r t i e s o f f a c t o r V M , c o m p a r a t i v e l y l i t t l e i s known r e g a r d i n g i t s i n t e r a c t i o n w i t h p u r i f i e d t i s s u e f a c t o r and t h e i n t e r a c t i o n o f f a c t o r X w i t h t h i s [ f a c t o r V l l - t i s s u e f a c t o r ] complex. The t i s s u e f a c t o r a p o p r o t e i n , t h e p r o t e i n component o f t h e t i s s u e f a c t o r l i p o p r o t e i n , h a s been i s o l a t e d f r o m s e v e r a l t i s s u e s . The most h i g h l y c h a r a c t e r i z e d a p o p r o t e i n p r e p a r a t i o n a p p e a r s t o be o b t a i n e d f r o m human b r a f n m i c r o s o m e s ( 2 3 ) , T i s s u e f a c t o r apop r o t e i n prepared i n t h i s study i s a s i n g l e - c h a i n glycoprotein c o n t a i n i n g a b o u t 6% c a r b o h y d r a t e and e x h i b i t e d a m o l e c u l a r w e i g h t o f 52,000 by SDS g e l e l e c t r o p h o r e s i s . The t i s s u e f a c t o r a p o p r o t e i n e x h i b i t s no c o a g u l a n t a c t i v i t y i n t h e a b s e n c e o f phospholipid. A d d i t i o n o f mixed p h o s p h o l i p i d s t o t h e a p o p r o t e i n , however, p a r t i a l l y restores the tissue factor coagulant a c t i v i t y . The r o l e o f t i s s u e f a c t o r a p p e a r s t o be s i m i l a r t o t h a t o f f a c t o r V I M i n t h a t i t s e r v e s t o enhance t h e l i m i t e d p r o t e o l y s i s o f f a c t o r X by f a c t o r V i l a s e v e r a l o r d e r s o f m a g n i t u d e . No e n z y m a t i c a c t i v i t y has been o b s e r v e d a s s o c i a t e d w i t h homogeneous preparations o f tissue factor. Moreover, i n c u b a t i o n o f pure bovine f a c t o r VII w i t h c r u d e , h i g h l y a c t i v e p r e p a r a t i o n s o f t i s s u e f a c t o r does n o t r e s u l t i n t h e f o r m a t i o n o f t w o - c h a i n , a c t i v a t e d f a c t o r V I I (21_). y
C.
Prothrombin A c t i v a t i o n
by A c t i v a t e d
Factor X
P r o t h r o m b i n i s c o n v e r t e d t o t h r o m b i n by f a c t o r Xa i n t h e p r e s e n c e o f c a l c i u m i o n s , p h o s p h o l i p i d and f a c t o r Va ( F i g u r e 3 ) . P r o t h r o m b i n and i t s mechanism o f a c t i v a t i o n has been i n t e n s i v e l y i n v e s t i g a t e d o v e r t h e l a s t two d e c a d e s , and t h e d e t a i l s o f t h i s complex r e a c t i o n a r e r e a s o n a b l y c l e a r (24). Prothrombin i s a g l y c o p r o t e i n with a molecular weight o f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
168
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
SURFACE HMW KININOGEN PREKALLIKREIN
FACTOR X I I
FACTOR
XII
FACTOR
IX
FACTOR CA
[FACTOR
2 +
,
IX
A
PLj FACTOR
IX -FACTOR 3
VIII
A
2 +
VIII -CA A
-PL]
Figure 1. Abbreviated mechanisms for the activation of factor X by the intrinsic pathway
FACTOR
XII
FACTOR
XI
; ]
; L
KALLIKREIN FACTOR
X
A
THROMBIN
FACTOR V I I
FACTOR ?
+
CA-
Figure 2. Abbreviated mechanisms for the activation of factor X by the extrinsic pathway
[FACTOR V I I
; 1
VII
-CA
T ISSUE 2 +
FACTOR
-TISSUE
FACTOR X
FACTOR X
; L
|
FACTOR
FACTOR]
X
1
:L
C A , PL j FACTOR V 2 +
;L
[FACTOR X^-FACTOR V ,-CA -PL] ;
2+
FACTOR XIII
PROTHROMBIN
Figure 3. Mechanisms for the formation of cross-linked fibrin following the activation of factor X
FIBRINOGEN j
2+
FACTOR XII I
A
FIBRIN (CROSS-LINKED)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
169
Coagulation
70,000. I t i s o n e o f t h e f o u r v i t a m i n K-dependent c l o t t i n g f a c t o r s a n d c o n t a i n s 10 y - c a r b o x y g l u t a m f c acid residues, I t s c o n c e n t r a t i o n i n plasma f a r exceeds t h a t o f any o t h e r c o a g u l a t i o n zymogen. The amino a c i d s e q u e n c e o f b o v i n e p r o t h r o m b i n h a s been d e t e r m i n e d (25) and t h e s e q u e n c e o f human p r o t h r o m b i n Is n e a r i n g completion. B o v i n e p r o t h r o m b i n c o n t a i n s 582 amino a c i d r e s i d u e s w i t h 12 d i s u l f i d e b r i d g e s . The t h r e e c a r b o h y d r a t e c h a i n s o f bovine prothrombin a r e N - g l y c o s f d i c a l l y l i n k e d t o asparagine r e s i d u e s a t p o s i t i o n s 7 7 , 101 and 376 ( 2 5 ) , The c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n o c c u r s a s a t w o s t e p p r o c e s s on t h e s u r f a c e o f a m a c r o m o l e c u l a r a g g r e g a t e commonly r e f e r r e d t o a s t h e " p r o t h r o m b i n a s e c o m p l e x . " T h i s c o m p l e x c o n s i s t s o f f a c t o r X a , f a c t o r V a , p h o s p h o l i p i d and c a l c i u m i o n s . P r e - t r e a t m e n t o f f a c t o r Xa w i t h DFP o b v i a t e s t h e c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n i n d i c a t i n g t h a t f a c t o r Xa i s t h e a c t i v e p r i n c i p a l i n t h i s complex f a c t o r , appears t o f u n c t i o cofactor in this reaction. Its a b i l i t y to potentiate this r e a c t i o n i s i n c r e a s e d s e v e r a l f o l d by p r e - t r e a t m e n t w i t h f a c t o r Xa or thrombin (26). The f i r s t s t e p i n t h e a c t i v a t i o n o f p r o t h r o m b i n i sthe cleavage o f a p e p t i d e bond between A r g - 2 7 4 and T h r - 2 7 5 by f a c t o r X a . T h i s r e a c t i o n r e l e a s e s a l a r g e I n a c t i v e g l y c o p e p t i d e fragment (mol wt 3 3 , 5 0 0 ) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f p r o t h r o m b i n and y i e l d s a s i n g l e - c h a i n t h r o m b o g e n i c p r o t e i n c a l l e d i n t e r m e d i a t e 11, T h i s p r o t e i n h a s no c o a g u l a n t a c t i v i t y a n d h a s a m o l e c u l a r w e i g h t of 39,000. I n t e r m e d i a t e II i s s u b s e q u e n t l y c l e a v e d by f a c t o r Xa a t a p e p t i d e bond between A r g - 3 2 3 a n d l l e - 3 2 4 t o y i e l d two-chain thrombin. T h r o m b i n I s composed o f a heavy c h a i n (mol wt 3 2 , 0 0 0 ) and a l i g h t c h a i n (mol wt 5 , 7 0 0 ) h e l d t o g e t h e r by a d i s u l f i d e bond. The heavy c h a i n o f t h r o m b i n a l s o c o n t a i n s t h e a c t i v e s i t e serine residue. D.
Formation
of Cross-linked
Fibrin
The c o n v e r s i o n o f f i b r i n o g e n t o f i b r i n monomer by t h r o m b i n I s t h e p e n u l t i m a t e s t e p i n t h e b l o o d c o a g u l a t i o n scheme. Fibrinogen i s a l a r g e g l y c o p r o t e i n (mol wt 3 ^ 0 , 0 0 0 ) composed o f t h r e e p a i r s of non-identical polypeptide chains. These c h a i n s , d e s i g n a t e d a , 8 and y , a r e h e l d t o g e t h e r by s e v e r a l d i s u l f i d e bonds. The m o l e c u l a r w e i g h t s f o r t h e a , 8 and y c h a i n s o f human f i b r i n o g e n a r e 6 3 , 5 0 0 , 5 6 , 0 0 0 and 4 7 , 0 0 0 , r e s p e c t i v e l y (2]_). T h u s , t h e s u b u n i t s t r u c t u r e f o r f i b r i n o g e n may be e x p r e s s e d a s a282Y2. During t h econversion o f f i b r i n o g e n t o f i b r i n , four s p e c i f i c a r g i n y l - g l y c i n e p e p t i d e bonds i n t h e a and 8 c h a i n s a r e c l e a v e d by t h r o m b i n w i t h t h e c o n c o m i t a n t r e l e a s e o f f o u r f i b r i n o p e p t i d e s . Two f i b r i n o p e p t i d e s A a r e r e l e a s e d f r o m t h e a m i n o - t e r m l n u s o f t h e a c h a i n s a n d two f i b r i n o p e p t i d e s B a r e r e l e a s e d f r o m t h e amino-terminal end o f t h e 8 c h a i n s . Subsequent t o t h e r e l e a s e o f t h e s e f i b r i n o p e p t I d e s , t h e f i b r i n monomers r a p i d l y a g g r e g a t e
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
170
GLYCOLIPIDS IN DISEASE PROCESSES
i n an o r d e r l y p r o c e s s t o f o r m t h e " s o l u b l e " f i b r i n p o l y m e r . This f i b r i n polymer i s s o l u b l e i n d e n a t u r a n t s such as urea o r g u a n i d i n e - h y d r o c h l o r i d e , and i s r e a d i l y a t t a c k e d by t h e f i b r i n o l y t i c protease, plasmin. C o n v e r s i o n o f t h e " s o l u b l e " f i b r i n p o l y m e r t o an " I n s o l u b l e " f i b r i n c l o t i s t h e terminal r e a c t i o n i n blood c o a g u l a t i o n . This r e a c t i o n i s c a t a l y z e d by a c t i v a t e d f a c t o r X I I I i n t h e p r e s e n c e o f calcium ions. In t h i s r e a c t i o n , i n t e r m o l e c u l a r p e p t i d e c r o s s l i n k a g e s a r e formed by f a c t o r XI I l a between an amino g r o u p f r o m a s p e c i f i c l y s i n e r e s i d u e on o n e f i b r i n monomer and a g l u t a m i n y l r e s i d u e o f an a d j a c e n t f i b r i n monomer ( 2 8 ) . Two e ( y - g l u t a m y l ) l y s i n e p e p t i d e bonds a r e r a p i d l y formed between i n t e r m o l e c u l a r y chains. Upon l o n g e r i n c u b a t i o n , c r o s s l i n k a g e s a r e a l s o o b s e r v e d between i n t e r m o l e c u l a r a c h a i n s . The t o t a l number o f c r o s s l i n k a g e s a p p e a r s t o be a b o u t s i x p e r mole o f f i b r i n , two of which i n v o l v e cross 1inkage w i t chain wit the a c h a i n s . The p r e s e n c f i b r i n polymer which i g q u i t e r e s i s t a n t t o plasmin d i g e s t i o n . F a c t o r XI I l a , t h e t r a n s g l u t a m i n a s e w h i c h c a t a l y z e s t h e c r o s s l i n k i n g r e a c t i o n s i n f i b r i n , c i r c u l a t e s i n plasma i n a p r e c u r s o r form. In t h e p r e s e n c e o f c a l c i u m i o n s , f a c t o r X I I I i s c o n v e r t e d by t h r o m b i n t o f a c t o r XI I l a by l i m i t e d p r o t e o l y s i s . F a c t o r X I I I i s a g l y c o p r o t e i n composed o f two A c h a i n s (mol wt 7 5 , 0 0 0 ) and two B c h a i n s (mol wt 8 8 , 0 0 0 ) . The n a t i v e p r o t e i n i s a t e t r a mer w i t h a m o l e c u l a r w e i g h t o f 3 2 0 , 0 0 0 ( 2 9 ) . Factor XIII i s a c t i v a t e d by t h r o m b i n i n a t w o - s t e p p r o c e s s . In t h e f i r s t s t e p , t h r o m b i n c l e a v e s an a r g i n y l - g l y c i n e bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e A c h a i n s , g i v i n g r i s e t o an i n a c t i v e t e t r a m e r i c i n t e r m e d i a t e and two p e p t i d e s (mol wt 4 , 0 0 0 ) . In t h e n e x t s t e p , which r e q u i r e s c a l c i u m i o n s , t h e t e t r a m e r i c intermediate d i s s o c i a t e s i n t o a c a t a l y t i c d i m e r ( A ' 2 ) and an i n a c t i v e d i m e r o f B chains (30). A l t h o u g h t h e A' c h a i n s a r e p r e s u m a b l y i d e n t i c a l , t h e a c t i v e c y s t e i n e r e s i d u e i s f o u n d i n o n l y one o f t h e c a t a l y t i c subunits. T h u s , f a c t o r XI I l a a p p e a r s t o be an e x a m p l e o f t h e " h a l f - o f - t h e - s i t e r e a c t i v i t y " enzymes ( 3 1 ) . E.
Mechanism lation
Involving P r o t e o l y s i s i n theTermination
o f Coagu-
Under normal p h y s i o l o g i c a l c o n d i t i o n s , t h e a c t i v a t i o n o f a zymogen t h r o u g h p r o t e o l y s i s i s e s s e n t i a l l y an i r r e v e r s i b l e p r o cess. A c c o r d i n g l y , s e v e r a l c o n t r o l mechanisms a r e a v a i l a b l e t o r e g u l a t e and t e r m i n a t e c o a g u l a t i o n f r o m s p r e a d i n g t h r o u g h o u t t h e circulation. P e r h a p s t h e p r i n c i p a l mechanism f o r t h e r e g u l a t i o n o f t h e a c t i v a t e d c o a g u l a t i o n f a c t o r s i s t h e i r n e u t r a l i z a t i o n by s e r i n e protease i n h i b i t o r s found i n t h e blood. Indeed, about 10% o f t h e p r o t e i n i n p l a s m a i s s e r i n e p r o t e a s e i n h i b i t o r s ( 3 2 ) . A n o t h e r i m p o r t a n t mechanism f o r t h e c o n t r o l o f c o a g u l a t i o n i n v o l v e s t h e i n a c t i v a t i o n o f s p e c i f i c c l o t t i n g f a c t o r s by l i m i t e d
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
Coagulation
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proteolysis. Although the d e t a i l s o f these reactions are j u s t b e g i n n i n g t o emerge, i t a p p e a r s t h a t t h r o m b i n i s c e n t r a l t o t h i s process. E x c e s s t h r o m b i n f o r m a t i o n i s c a r e f u l l y c o n t r o l l e d by several negative feedback r e a c t i o n s e i t h e r i n i t i a t e d o r d i r e c t l y c a t a l y z e d by t h r o m b i n . As p o i n t e d o u t e a r l i e r , p r o t e o l y t i c m o d i f i c a t i o n o f f a c t o r V and f a c t o r VI If by t h r o m b i n e n c h a n c e s t h e coagulant a c t i v i t y o f these p r o t e i n s several f o l d . Continued i n c u b a t i o n o f these p r o t e i n s w i t h t h r o m b i n , however, r e s u l t s i n t h e loss o f t h e i r coagulant a c t i v i t y (15,33). Thrombin a l s o c a t a l y z e s t h e r a p i d c l e a v a g e o f a p e p t i d e bond between A r g - 1 5 6 and Ser-157 i n prothrombin g i v i n g r i s e t o a non-thrombogenic fragment (mol wt 23,000) and a t h r o m b o g e n i c i n t e r m e d i a t e I (mol wt 5 5 , 0 0 0 ) . Intermediate I i s loosely associated with the "prothrombinase c o m p l e x " and c o n s e q u e n t l y c l e a v e d by f a c t o r Xa a t a much s l o w e r r a t e than p r o t h r o m b i n . Hence, t h e o v e r a l l e f f e c t i s t o reduce thrombin f o r m a t i o n as a R e c e n t l y , a new v i t a m i c o v e r e d and t e n t a t i v e l y i d e n t i f i e d a s P r o t e i n C ( J 4 J , Protein C i s a g l y c o p r o t e i n composed o f a heavy c h a i n (mol wt 40,000) and a l i g h t c h a i n (mol wt 22,000) h e l d t o g e t h e r by d i s u l f i d e bonds. Incubation o f P r o t e i n C with thrombin r e s u l t s i n the cleavage o f a s i n g l e p e p t i d e bond between A r g - 1 4 and l l e - 1 5 i n t h e heavy chain w i t h the concomitant formation o f a c t i v a t e d P r o t e i n C (35). A c t i v a t e d P r o t e i n C i s a s e r i n e p r o t e a s e and t h e a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h e heavy c h a i n . The amino a c i d sequence s u r r o u n d i n g t h e a c t i v e s i t e s e r i n e r e s i d u e i n a c t i v a t e d P r o t e i n C i s homologous t o t h e a c t i v e s i t e r e g i o n s o f t h e s e r i n e proteases involved i n blood coagulation. Activated Protein C m a r k e d l y p r o l o n g s t h e c l o t t i n g t i m e o f p l a s m a , and t h i s a n t i c o a g u l a n t e f f e c t was t o t a l l y o b v i a t e d by p r i o r i n c u b a t i o n o f t h e enzyme w i t h DFP. R e c e n t e v i d e n c e i n d i c a t e s t h a t a c t i v a t e d P r o t e i n C, i n t h e p r e s e n c e o f c a l c i u m i o n s and p h o s p h o l i p i d , i s a p o t e n t i n a c t i v a t o r o f f a c t o r V and f a c t o r V I M ( 3 5 , 3 6 ) » and t h a t t h e s e i n a c t i v a t i o n r e a c t i o n s were a b s o l u t e l y d e p e n d e n t on t h e e n z y m a t i c a c t i v i t y o f a c t i v a t e d P r o t e i n C. T h u s , i t a p p e a r s t h a t the l e v e l o f t h r o m b i n a c t i v i t y i s c a r e f u l l y r e g u l a t e d by n e g a t i v e feedback r e a c t i o n s which i n v o l v e the l i m i t e d p r o t e o l y s i s o f prot h r o m b i n , f a c t o r V and f a c t o r V I M e i t h e r d i r e c t l y c a t a l y z e d by thrombin o r i n d i r e c t l y through the formation o f a c t i v a t e d P r o t e i n C.
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Griffin, J . H., and Cochrane, C. G. (1976) Methods Enzymol. 45, 56-65. Fujikawa, K., Walsh, K. A . , and Davie, E. W. (1977) Biochemistry 16, 2270-2278, Fujikawa, K., Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 4182-4188.
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Chan, J . Y. C., Habal, K. M., Burrowes, C. E . , and Movat, H. Z. (1976) Thromb. Res. 9, 423-430. Wiggins, R. C., Bouma, B. N . , Cochrane, C. G., and Griffin, J. H. (1977) Proc. Natl. Acad. Sci. USA 74, 4636-4640. Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 58315839.
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DiScipio, R. G., Hermodson, M. A . , Yates, S. G., and Davie, E. W. (1977) Biochemistry 16, 698-706. Fujikawa, K., Thompson, A. R., Legaz, M. E . , Meyer, R. G., and Davie, E. W. (1973) Biochemistry 12, 4938-4945. Stenflo, J., Fernlund, P., Egan, W., and Roepstorff, P. (1974) Proc. Natl. Acad. Sci. USA 71, 2730-2733. Furie, B. C., Mann, K. G., and Furie, B. (1976) J. Biol. Chem. 251, 3235-3241
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Fujikawa, K., Legaz Biochemistry 13, 4508-4516 McKee, P. A . , Anderson, J . C., and Switzer, M. E. (1975) Ann. N.Y. Acad. Sci. 240, 8-33. Legaz, M. E . , Weinstein, M. J., Heldebrant, C. M., and Davie, E. W. (1975) Ann.N.Y.Acad. Sci. 240, 43-61. Vehar, G. A . , and Davie, E.W.(1977)Science 197, 374-376. Titani, K., Fujikawa, K., Enfield, D. L., Ericsson, L. H . , Walsh, K. A . , and Neurath, H. (1975) Proc. Natl. Acad. S c i . USA 72, 3082-3086.
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K i s i e l , W., Fujikawa, K., and Davie, E. W. (1977) Biochemistry 16, 4189-4194. Kisiel, W., unpublished data. Radcliffe, R., and Nemerson, Y. (1976) J. Biol. Chem. 251 4797-4802.
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Schwartz, M. L., Pizzo, S, V., Hill, R, C., and McKee, P. A. (1973) J. Biol. Chem. 248, 1395-1407. Chung, S. I . , Lewis, M. C., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950. Levitzki, Α., Stallcup, W. Β., and Koshland, D. E. (1971) Biochemistry 10, 3371-3378. Heimburger, Ν., Haupt, Η., and Schwick, H. (1971) in Proc. Int. Res. Conf. Proteinase Inhibitors, eds. Fritz, H., and Tschesche, H. (W. de Gruyter, Berlin), pp. 1-21. Colman, R. W. (1969) Biochemistry 8, 1438-1445. Stenflo, J . (1976) J. Biol. Chem. 251, 355-363. Kisiel, W., Canfield, W. Μ., Ericsson, L. H., and Davie, E. W. (1977) Biochemistry 16, 5824-5831. Vehar, G., unpublished results.
RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10 Biosynthesis of Murine Leukemia Virus Membrane Proteins and their Assembly into Virus Particles R. B. ARLINGHAUS, E. C. MURPHY, JR., R. B. NASO, L. J. ARCEMENT, and W. L. KARSHIN Biology Department, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030
The cellular plasma membrane forms the surface layer of C-type RNA tumor virus particles. The c e l l ' s plasma membrane is also involved in one of the important phases of the l i f e cycle of RNA tumor viruses, that is the budding of the complete virus particles from the infected c e l l . C-type RNA tumor viruses contain a core structure surrounded by a l i p i d envelope through which spikes protrude. These spikes are believed to be composed, in mouse systems, of the v i r a l glycoprotein gp69/71 (1). Two non -glycosylated proteins termed p12(E) and p15(E) are also associated with the virion envelope. Their roles are not definitely known but recent studies suggest that gp69/71 and p15(E) exist as a complex and this complex forms the basic unit of the protruding spike (1, 2, 3). The core of these viruses is composed of four internal proteins termed p30, p15, pp12 (phosphoprotein) and p10 (4). The latter two have been shown to be associated with the v i r a l genomic RNA (5, 6). Viral p30 is thought to be the capsid protein of the core (4) whereas v i r a l protein p15 (a hydrophobic protein) could be involved in the association of the core with the l i p i d envelope. The virus also contains a DNA polymerase in its core. This polymerase can readily copy RNA into DNA with an appropriate p r i mer. It is present in virions in amounts estimated to be 50-100 molecules per virus particle (7) whereas the internal structural proteins are thought to be present in approximately 5,000 copies per virion (8). The envelope proteins [gp69/71, p15(E)] are also initially present in relatively large amounts but gp69/71 appears to be lost from the virion probably because of proteolytic degradation that releases the mature glycoprotein into the surrounding medium (9). 0-8412-0452-7/78/47-080-180$05.25/0
© 1978 American Chemical Society
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These v i r a l p r o t e i n s are encoded by three d i f f e r e n t genes w i t h i n the 35S (-3 x 10^ daltons o r 10,000 bases) v i r a l genome. Each v i r a l p a r t i c l e i s thought to c o n t a i n two i d e n t i c a l copies of the RNA genome. The gene coding f o r the four i n t e r n a l core prot e i n s has been termed g a g o r group antigens; that coding f o r the v i r a l RNA-directed DNA polymerase i s termed p o l o r reverse t r a n s c r i p t a s e (RT); the envelope p r o t e i n s are coded w i t h i n the envelope gene or e n v (10). The purpose o f t h i s r e p o r t i s to summarize the events l e a d ing to the formation of the mature v i r a l envelope p r o t e i n s and t h e i r assembly i n t o v i r i o n s . We a l s o w i l l propose a model f o r v i r i o n assembly and budding of the v i r u s p a r t i c l e from the c e l l . f
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The V i r i o n P r o t e i n s of Rauscher Murine Leukemia V i r u s Rauscher murine leukemia v i r u s (R-MuLV) coded p r o t e i n s can be r e s o l v e d by e l e c t r o p h o r e s i denaturing c o n d i t i o n s ( F i g can be seen are p30, p l 5 , pl2(E) and plO; minor amounts o f gp69/71 and pl5(E) are a l s o present i n the v i r i o n . A s u b s t a n t i a l amount o f an uncleaved precursor to the core p r o t e i n s , termed Pr65§ S, i s a l s o present i n v i r u s . The v i r a l phosphoprotein ppl2 i s not s o l u b i l i z e d by t h i s procedure but i t i s r e a d i l y seen i n v i r u s denatured w i t h 6 M quanidine-HCl and f r a c t i o n a t e d by molecu l a r s e i v e chromatography i n 6 M guanidine. The r a d i o a c t i v e v i r u s , whose p r o t e i n s are sljown i n F i g . 1, was prepared by i n c u b a t i n g i n f e c t e d c e l l s w i t h ^ C-arginine and l y s i n e f o r a 48 hr i n t e r v a l . Under these c o n d i t i o n s much of the v i r a l gp69/71 i s l o s t from the v i r u s p a r t i c l e (9). This l o s s of v i r a l gp69/71 may correspond to the low amount of pl5(E) that i s present i n mature v i r u s p a r t i c l e s (see below). Murine C-type v i r u s e s appear to c o n t a i n a complex made up of gp69/71 and pl5(E) (1, 2, 3). The gp69/71-pl5(E) complex can be d i s s o c i a t e d w i t h s u l f h y d r i l reagents such as mercaptoethanol (1, _2, 3) . This complex may be the b a s i c u n i t of the surface s t r u c ture of the v i r u s . V i r a l gp69/71 has been shown to be on the surface of the v i r u s by s e v e r a l methods. One i n v o l v e s l a c t o peroxidase-catalyzed i o d i n a t i o n of the v i r i o n s u r f a c e components (11, 12). Another i n v o l v e s sodium borohydride r e d u c t i o n f o l l o w ing galactose oxidase treatment (13). A t h i r d procedure concerns the use of immunoelectromicroscopy using monospecific a n t i s e r a r a i s e d against p u r i f i e d gp70 (14), and most important, anti-gp70 can n e u t r a l i z e i n f e c t i v i t y (15, 16). The surface l o c a t i o n o f pl5(E) i s i n d i c a t e d by the f i n d i n g that a n t i s e r a to pl5(E) can p r e c i p i t a t e i n t a c t v i r u s (17, 18, 19) u n l i k e a n t i s e r a prepared against v i r a l i n t e r n a l components. Thus, i t i s q u i t e evident that both pl5(E) and gp69/71 are present i n the same place i n the v i r i o n which i s c o n s i s t e n t w i t h t h e i r being complexed together. a
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Figure
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Preparative sodium dodecyl sulfate-polyacrylamide mature viral proteins
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Rauscher murine leukemia virus (R-MuLV) was grown in a Balh/C spleen-thymus cell line (JLS-V5 cells) in the presence of C-arginine and lysine for 48 hr. Virus was purified as described (30), and viral proteins were separated by electrophoresis on a 11.25% polyacrylamide slab gel. The radioactive virus was distributed among the three sample wells after boiling in electrophoresis sample buffer (20). The radioactive bands were detected by fluorography using pre-flashed x-ray film so as to obtain a linear response to the amount of radiactivity (26). 14
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I n t r a c e l l u l a r V i r a l S p e c i f i c Glycoproteins V i r u s i n f e c t e d and u n i n f e c t e d NIH Swiss mouse embryo f i b r o b l a s t s (JLS-V16 c e l l s ) were incubated f o r 5-1/2 hr w i t h C glucosamine to detect v i r a l g l y c o p r o t e i n s . The cytoplasmic ext r a c t , t r e a t e d w i t h NP-40 and sodium deoxycholate detergents (20), was mixed w i t h an optimal c o n c e n t r a t i o n of antiserum r a i s e d i n r a b b i t s against p u r i f i e d R-MuLV that had been d i s r u p t e d by the above detergents (21). Figure 2 shows the r e s u l t s of f r a c t i o n a t i o n of the immunoprecipitates on a 10% sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l . Figure 2 lane A shows the r a d i o a c t i v e g l y c o p r o t e i n s were not observed i n u n i n f e c t e d c e l l s i n the area where two broad bands termed g P r 9 0 and gp69/71 were seen i n i n f e c t e d c e l l s ( F i g . 2, lane B). These r e s u l t s i n d i c a t e that two major g l y c o p r o t e i n s are present i n v i r a l i n f e c t e d c e l l s but not i n u n i n f e c t e d c e l l s . One of the g l y c o p r o t e i n s corresponds i n molecular weight to the other we have shown to b gp69/71 and the non-glycosylated p r o t e i n pl5(E) (see below and references 22, 23). Since both gp69/71 and g P r 9 0 r e a d i l y incorporate radioact i v e glucosamine, i t was o f i n t e r e s t to t e s t r a d i o a c t i v e l a b e l i n g w i t h other sugars. Of p a r t i c u l a r i n t e r e s t i s fucose s i n c e i t i s o f t e n a t e r m i n a l sugar i n g l y c o p r o t e i n s (24). Fucosyl tranferases are a l s o present i n plasma membranes o f the c e l l (25) thereby r e s t r i c t i n g f u c o s y l a t i o n to the l a t t e r stages of g l y c o p r o t e i n proc e s s i n g . With these p o i n t s i n mind, we then incubated i n f e c t e d c e l l s w i t h % - l a b e l e d fucose and p r e c i p i t a t e d the v i r a l glycoprot e i n s e i t h e r w i t h anti-RLV serum ( F i g . 2, lane C) or anti-gp69/71 ( F i g . 2, lane D). The r e s u l t s showed that g P r 9 0 d i d not i n corporate fucose whereas the mature v i r a l g l y c o p r o t e i n (gp69/71) d i d . Lanes G and H of F i g . 2 a l s o show that v i r i o n gp69/71 a l s o i n c o r p o r a t e s r a d i o a c t i v e fucose as w e l l as glucosamine. Thus, gPr90 appears to c o n t a i n glucosamine but l a c k s fucose and d i s t i n g u i s h e s i t c l e a r l y from the mature gp69/71. In a p a r a l l e l experiment v i r u s - i n f e c t e d c e l l s were incubated w i t h r a d i o a c t i v e glucosamine f o r 5-1/2 hr and the cytoplasmic ext r a c t was d i v i d e d i n t o two p a r t s . One p o r t i o n was immunoprecipit a t e d w i t h anti-gp69/71 ( F i g . 2, lane E) and the other p o r t i o n was i s o l a t e d by anti-R-MuLV immunoprecipitation ( F i g . 2, lane F ) . Again the major v i r a l glucosamine-containing g l y c o p r o t e i n s recogn i z e d bv e i t h e r sera were gp69/71 and g P r 9 0 . We note that n ti~R-MuLV sera a l s o p r e c i p i t a t e d a minor glucosamine-containing g l y c o p r o t e i n that migrates j u s t slower than g P r 9 0 ( F i g . 2, lanes B and F ) . This g l y c o p r o t e i n may be r e l a t e d to gag' gene products because i t i s not recognized by anti-gp69/71 but i t i s seen w i t h anti-RLV. Anti-RLV serum contains a n t i b o d i e s t o g a g and 'env' determinants but not to ' p o l determinants (26). I n f r e q u e n t l y another p r o t e i n was a l s o detected i n the anti-R-MuLV p r e c i p i t a t e from glucosamine l a b e l e d i n f e c t e d c e l l s . I t comi1 4
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grates w i t h Pr2008 S~P°l which i s the precursor to the v i r i o n r e verse t r a n s c r i p t a s e (26, 27, 28). Kabat and co-workers have r e ported t h a t F r i e n d leukemia v i r u s i n f e c t e d c e l l s c o n t a i n 'gag gene p r o d u c t - r e l a t e d g l y c o p r o t e i n s of 95,000 and 200,000 mol. wt. (29). The l a t t e r were found i n a d d i t i o n to the v i r a l 'env' gener e l a t e d g l y c o p r o t e i n s . The r o l e of the 'gag' gene-related g l y c o p r o t e i n s i n the v i r u s l i f e c y c l e i s unknown. To show the s p e c i f i c i t y of immunoprecipitation, R-MuLV i n f e c t e d c e l l s were p u l s e - l a b e l e d w i t h -^C-amino a c i d s f o r 10 min and p a r a l l e l c u l t u r e s were p u l s e - l a b e l e d f o r 10 min and then i n cubated i n complete growth medium f o r 60 min (chase). Cytoplasmic e x t r a c t s were p r e c i p i t a t e d w i t h monospecific antiserum d i r e c t e d a g a i n s t R-MuLV gp69/71 and p30 (22). Anti-gp69/71 p r e c i p i tated g P r 9 0 almost e x c l u s i v e l y i n p u l s e - l a b e l e d c e l l s ( F i g . 3, lane A) and both g P r 9 0 and gp69/71 i n chase incubated c e l l s ( F i g . 3, lane B). Anti-p30 serum, however, d i d not recognize gPr90 i n pulse-labele mainly precursors Pr80g A f t e r chase i n c u b a t i o n ( F i g . 3, lane D), a n a l y s i s of the anti-p30 p r e c i p i t a t e showed t h a t Pr80S § disappeared and P r 6 5 was r e duced i n i t s i n t e n s i t y w h i l e p30 appeared as a major band. No gp69/71 or g P r 9 0 were p r e c i p i t a t e d by a n t i s e r a d i r e c t e d a g a i n s t 30. The k i n e t i c s of v i r a l g l y c o p r o t e i n s y n t h e s i s were s t u d i e d by p u l s e - l a b e l i n g c e l l s w i t h -^C-amino a c i d s and chasing r e p l i c a t e c u l t u r e s f o r 2, 4 and 6 hr p e r i o d s . The r e s u l t s c l e a r l y showed that gp69/71 was not l a b e l e d i n a 10 min p u l s e - l a b e l i n g ( F i g . 4, lane B) but became r a d i o a c t i v e between 60 and 120 min of the chase i n c u b a t i o n ( F i g . 4, lane E) w h i l e a t the same time there was a s l i g h t decrease i n the i n t e n s i t y of g P r 9 0 . Chase incubat i o n s of up to 6 hr s t i l l showed a s i g n i f i c a n t amount of g P r 9 0 v but the r a t i o of gp69/71 to g P r 9 0 reached a value greater than one whereas a f t e r 2 hr of chase i t was l e s s than one. As p r e v i o u s l y r e p o r t e d , p r e c i p i t a t i o n of c e l l u l a r e x t r a c t s w i t h antiserum to d i s r u p t e d v i r u s revealed other v i r a l p r o t e i n s (e.g. p30 and p l 5 ) t h a t appeared upon chase at the expense of Pr80gag and Pr65§ 8 (20, 21, 2_2, 30) ( F i g . 4, lanes A, D, G, and J ) . V i r a l pl2(E) a l s o appeared during long chase periods (2 to 6 hr) whereas only pl5(E) was seen i n short chase periods (1 h r ) . 1
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Presence of gp69/71 and p!5(E) Peptide Sequences i n g P r 9 Q e n V
e n V
We have p r e v i o u s l y shown t h a t g P r 9 0 and gp69/71 c o n t a i n many t r y p t i c peptides that comigrate on i o n exchange columns (22). However, the p a t t e r n s obtained were q u i t e complex, to s i m p l i f y the complexity of the t r y p t i c d i g e s t p r o f i l e s and the subsequent comparisons of the peptide sequences, we e l e c t e d to tag the v i r a l p r o t e i n s w i t h r a d i o a c t i v e t y r o s i n e (23, 30). F i g u r e 5 shows i o n exchange p r o f i l e s i n which l ^ C - t y r o s i n e - l a b e l e d g P r 9 0 tryptic peptides were mixed w i t h t r y p t i c peptides of % - t y r o s i n e l a b e l e d e n v
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ARLINGHAUS E T A L .
Figure 2.
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Pohjacrylamide gel electrophoresis of R-MuLV glycoproteins from infected cells and virus.
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specific
Cytoplasmic extracts from uninfected (lane A) and R-MuLV-infected NIH Swiss mouse embryo fibroblasts (JLS-V16 cells) (lane B) were labeled for 5V2 hr with C-glucosamine and the anti-R-MuLV immunoprecipitates were analyzed as in Figure 1. Cytoplasmic extracts from R-MuLV-infected JLS-V15 cells were labeled with H-fucose were immunoprecipitated with anti-gp69/71 serum (lane C) or anti-R-MuLV serum (lane D). R-MuLVinfected cells were labeled for 5 /2 hr with C-glucosamine and the antigp69/71 immunoprecipitate (lane E) or the anti-R-MuLV immunoprecipitate (lane F) also were analyzed. Columns G and H are H-fucose and C-glucosa7nine-labeled virus, respectively, obtained form growth medium from R-MuLV-infected JLS-V16 cells following a SV2 hr labeling period. 14
3
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Figure 3. Analysis of intracellular RMuLV specific proteins precipitated by anti-gp69/71 and anti-p30 sera. Cytoplasmic extracts from R-MuLV-infected JLS-V16 cells labeled with C-labeled amino acids were prepared and immunoprecipitation was performed with either anti-gp69/71 or anti-p30 serum. Electrophoresis on 10% gels andfluorographywere as in Figure 1. Column A, anti-gp69/71 immune precipitate from a 10-min pulse labeling; column B, antigp69/71 immune precipitate from a 10-min pulse labeling with a 60 min chase; column C, anti-p30 immune precipitate from a 10min pulse labeling; column D, anti-p30 immune precipitate of a 10-min pulse labeling with a 60-min chase incubation. 14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 4. Formation chase experiment Cytoplasmic extracts from infected JLS-V16 cells were prepared, and immunoprecipitation was performed as described. Electrophoresis on 6-12% gradient gels and fluorography were carried out as in Figure 1. Columns Vi are C-labeled amino acid marker virus; columns A, B, and C, 10 min pulse with Clabeled amino acid protein hydrolysate; columns D, £, and F, 2-hr chase incubation of the 10-min pulse labeling; columns G, 77, and 7, 4-hr chase; columns J, K, and L, 6-hr chase; columns A, D, G, and J, anti-R-MuLV precipitated; columns B, E, 77, and K, anti-gp69/71 precipitated; and columns C, F, 7, and L, precipitated with anti-MuLV absorbed with excess R-MuLV proteins. 14
14
Figure 5. Ion exchange chromatography of tryptic digests of tyrosine-labeled gFr90 and gp69/71. The arrows show the two additional tyrosine-containing tryptic peptides in gPr90 that are not found in gp69/71. €nv
env
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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gp69/71. The r e s u l t s c l e a r l y show that g P r 9 0 shares many t y r o s i n e - c o n t a i n i n g t r y p t i c peptides w i t h gp69/71. Figure 5 a l s o shows that there are at l e a s t two a d d i t i o n a l t y r o s i n e - c o n t a i n i n g t r y p t i c peptides i n g P r 9 0 t h a t are not found i n gp69/71 (see arrows i n F i g . 5). An a n a l y s i s of a -*- C-tyrosine-labeled pl5(E) t r y p t i c d i g e s t mixed w i t h a ^ H - t y r o s i n e - l a b e l e d g P r 9 0 digest ( F i g . 6) c l e a r l y showed that these two a d d i t i o n a l t r y p t i c peptide f r a c t i o n s are d e r i v e d from p l 5 ( E ) . We conclude from these r e s u l t s that pl5(E) and gp69/71 are cleavage products o f g P r 9 0 . e n v
e n v
4
e n v
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The Gene Order of the P r o t e i n s W i t h i n g P r 9 Q
e n V
e t l V
The order of gp69/71 and pl5(E) w i t h i n g P r 9 0 was examined by i n c u b a t i n g i n f e c t e d c e l l s w i t h r a d i o a c t i v e amino a c i d s i n the presence and absence of pactamycin, a drug that s e l e c t i v e l y i n h i b i t s the i n i t i a t i o n of t r a n s l a t i o n at 5 x 10""^ M but not e l o n g a t i o n (31, 32). In th t i a l l y l a b e l the C-termina In the case of g P r 9 0 , we could determine which p r o t e i n i s a t the C-terminus. R e s u l t s of such s t u d i e s (23) showed that pactamycin treatment f o r 30 to 60 seconds before and during p u l s e l a b e l i n g reduced the amount of r a d i o a c t i v e gp69/71 formation i n a chase i n c u b a t i o n , whereas r a d i o a c t i v e pl5(E) was increased r e l a t i v e to that found i n the absence of the drug. The r e s u l t s were q u a n t i t a t e d y i e l d i n g a r a t i o of pl5(E) to gp69/71 of 0.39 i n the c o n t r o l and 0.76 i n the presence of pactamycin. These r e s u l t s suggest that pl5(E) i s C-terminal i n g P r 9 0 . e n v
fenv
V i r a l P r o t e i n p!2(E) We have i d e n t i f i e d a new v i r a l p r o t e i n , termed p l 2 ( E ) , i n v i r u s p a r t i c l e s . I t can be d i s t i n g u i s h e d from phosphoprotein p l 2 (ppl2) on s e v e r a l grounds. F i r s t pl2(E) i s not phosphorylated (23), i t migrates i n the v o i d volume f r a c t i o n of a 6 M guanidineHC1 column whereas ppl2 e l u t e s i n the 12,000 mol. wt. range (23), and t h i r d l y i t has a peptide map much d i f f e r e n t from ppl2 (23); and l a s t l y , pl2(E) and ppl2 can be r e s o l v e d by 2-dimensional g e l s c o n s i s t i n g of i s o e l e c t r i c f o c u s i n g i n the f i r s t dimension and sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l e l e c t r o p h o r e s i s (PAGE) i n the second (23). We emphasize the p o i n t that i s o l a t i o n of pl2(E) from v i r u s by SDS-PAGE o f t e n y i e l d s p l 2 ( E ) contaminated w i t h fragments of p l 5 and p30. We have found i t necessary to use a two-step procedure i n v o l v i n g guanidine-HCl agarose column chromatography f o l l o w e d by SDS-PAGE i n order to o b t a i n a r e l a t i v e l y pure p r e p a r a t i o n of p l 2 ( E ) . The r o l e of pl2(E) i n v i r u s s t r u c t u r e i s not known. I t i s tempting to propose that i t i s a degradation product of pl5(E) and i s the r e s u l t of a p r o t e o l y t i c cleavage of the gp69/71-pl5(E) complex r e s u l t i n g i n the l o s s o f gp69/71 from the v i r i o n (4, 9) and the generation of an uncomplexed pl2(E) remaining i n the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
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Figure 6. Ion exchange chromatography of tryptic digests of tyrosine-labeled gPr90 and pl5(E). The pl5(E) used in this experiment was purified from virus by sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described in the legend to Figure 1. env
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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v i r u s envelope. We propose that t h i s p r o t e o l y t i c cleavage occurs a f t e r the v i r u s i s r e l e a s e d from the c e l l i n t o the c u l t u r e medium. We have shown t h a t as R-MuLV p a r t i c l e s age i n the c u l t u r e medium i n which they were r e l e a s e d , such p a r t i c l e s show a steady l o s s o f gp69/71 (Arlinghaus, unpublished r e s u l t s ) . C e l l - F r e e Synthesis of an Unglycosylated P15(E)
Precursor t o gp69/71 and
C e l l - f r e e t r a n s l a t i o n o f genomic RNAs from murine and avian RNA tumor v i r u s e s has shown that the f u l l - l e n g t h 35S RNA can code f o r the core p r o t e i n precursor P r 6 5 S 8 (28, 33, 34, 35. 36, 37) as w e l l as f o r a l a r g e r p o l y p r o t e i n c o n t a i n i n g both 'gag' and p o l gene products (_28, 34, 3 9 ) . The e n v gene product has never been observed to be coded f o r by f u l l l e n g t h v i r i o n genomic RNA although the i n f o r m a t i o n f o r t h i s gene product i s d e f i n i t e l y encoded i n th In order t o determin could be synthesized from i n t r a c e l l u l a r RNA from v i r a l i n f e c t e d c e l l s , we i s o l a t e d 18-25S RNA and 25-35S RNA from a t o t a l RNA ext r a c t by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . These RNA c l a s s e s were again f r a c t i o n a t e d on separate sucrose g r a d i e n t s and i n d i v i d u a l f r a c t i o n s were analyzed i n a mRNA-dependent c e l l - f r e e amino a c i d i n c o r p o r a t i o n system (28^, 34) . The r e s u l t s o f t h i s experiment, d i s p l a y e d i n F i g . 7 (lanes E and F) showed that only RNA of about 20-22S i n s i z e , can be t r a n s l a t e d i n t o a polypeptide prec i p i t a b l e by antiserum to gp69/71. This polypeptide had an apparent molecular weight of 68,000 to 70,000. I n some experiments t h i s polypeptide migrated as two bands (a d o u b l e t ) . Polypeptides both l a r g e r and s m a l l e r than the 68,000 t o 70,000 were a l s o recogn i z e d by anti-gp69/71 serum. However, the smaller polypeptides were found i n a l l RNA f r a c t i o n s and the l a r g e r ones were found i n c o n s i d e r a b l y s m a l l e r q u a n t i t i e s than the 68,000 t o 70,000 mol. wt. polypeptide. The i d e n t i t y o f these s m a l l e r and l a r g e r polypept i d e s i s unknown. Immunoprecipitation o f the c e l l - f r e e t r a n s l a t i o n product of the 25S t o 35S RNA c l a s s w i t h anti-gp69/71 i n d i cated t h a t the 68,000 t o 70,000 mol. wt. polypeptide was absent ( r e s u l t s not shown). Hence, a 20-22S RNA seems to be the s o l e mRNA f o r an envelope-related p r o t e i n i n R-MuLV i n f e c t e d c e l l s . The t r a n s l a t i o n product o f each o f the RNA f r a c t i o n s i n the 18-25S and 25-30S s i z e c l a s s e s was a l s o t e s t e d by immunoprecipit a t i o n w i t h anti-p30 serum. The r e s u l t s showed that the g a g o r core p r o t e i n precursor Pr658 § and the RT precursor Pr200§ 8"P are synthesized e x c l u s i v e l y from RNA which i s greater than 28S i n s i z e ( r e s u l t s not shown). T r y p t i c peptide a n a l y s i s o f t h i s 65,000 mol. wt. product showed that i t had methionine-containing t r y p t i c peptides c h r a c t e r i s t i c of a u t h e n t i c P r 6 5 ^ ^ . We have p r e v i o u s l y e s t a b l i s h e d that P r 6 5 8 8 i s the major product and Pr2008 8"" i s a minor product i n c e l l - f r e e p r o t e i n s y n t h e s i z i n g systems o f 35S R-MuLV genomic RNA t r a n s l a t e d (34). a
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In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Figure 7. Sodium dodecyl sulfate slab gel electrophoresis of the cell-free product produced from 20-22S mRNA from R-MuLV infected JLS-V5 cells. Total cellular nucleic acid was extracted from RMuLV-infected JLS-V5 cells as described (28), and RNA was selectively obtained by washing the nucleic acid alcohol precipitate with 3M LiClSmM EDTA = (pH 6) which removes DNA.The RNA was fractionated by velocity sedimentation on a sucrose gradient, and the 18S-to-25S RNA fraction was pooled and again separated on a sucrose gradient. Aliquots of the fractions (1 ^g of RNA) were analyzed in a mRNA-dependent protein synthesizing system (28,34). The protein products were immunoprecipitated with anti-gp69/71 serum and analyzed on a 6-12% gradient slab-gel of polyacrylamide, as in Figure 1. Lane A is a pulse-chase experiment from R-MuLV-infected JLS-V16 cells showing authentic viral specific protein precursors obtained with anti-R-MuLV serum (20). Lanes B-G represent aliquots across a gradient in which 18S ribosomal RNA sediments to the middle of the centrifuge tube (lane D). Lane B represents the top two fractions, lane G represents the bottom two fractions, and fractions C, D, E, and F represent the in-between fractions.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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ARLINGHAUS E T A L .
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Peptide Maps o f the Envelope-related P r o t e i n Synthesized From I n t r a c e l l u l a r 20-22S mRNA I f the 68,000 t o 70,000 mol. wt. polypeptide coded f o r by 20-22S mRNA i s r e l a t e d t o R-MuLV envelope precursor g P r 9 0 , i t should possess t r y p t i c peptides c h a r a c t e r i s t i c o f gp69/71 and p l 5 ( E ) . Therefore, 3 5 - e t h i o n i n e - l a b e l e d 68,000 t o 70,000 mol. wt. p o l y p e p t i d e product of 20-22S mRNA was digested w i t h t r y p s i n and mixed w i t h a u t h e n t i c g P r 9 0 t r y p t i c peptides l a b e l e d w i t h 3 -methionine. The mixture was f r a c t i o n a t e d by i o n exchange chromatography ( F i g . 8 ) . A n e a r l y p e r f e c t c o - e l u t i o n of t r y p t i c methionine-containing peptide was observed. The one exception being a minor peak ( F r a c t i o n 45) i n the i n v i t r o synthesized product that was n o t observed i n the a u t h e n t i c g P r 9 0 . The pept i d e peak e l u t i n g a t F r a c t i o n 76 i s c h a r a c t e r i s t i c of pl5(E) (20, 22) as i s the peptide peak a t F r a c t i o n 83 i f the t r y p t i c peptides are not o x i d i z e d w i t h performi c h a r a c t e r i s t i c o f gp69/7 s u l t s ) . We conclude from these experiments that the 68,000 t o 70,000 mol. wt. p o l y p e p t i d e termed P r 6 8 , i s i d e n t i c a l t o gPr90 i n o v e r a l l peptide content and P r 6 8 probably r e p r e sents the u n g l y c o s y l a t e d form of that molecule. e n v
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Since a 20-22S i n t r a c e l l u l a r mRNA species codes f o r P r 6 8 , i t was o f i n t e r e s t to determine whether RNA from v i r u s p a r t i c l e s had a s i m i l a r coding c a p a c i t y . V i r a l RNA p r e p a r a t i o n s obtained from v i r u s , t h a t has remained 24 h r i n the c u l t u r e f l u i d , have i n a d d i t i o n t o 35S RNA a range o f fragments o f the f u l l l e n g t h genome (35S) whose sedimentation r a t e v a r i e s down to 4S. T r a n s l a t i o n of v a r i o u s s i z e c l a s s e s o f v i r i o n RNA f r a c t i o n a t e d on a sucrose grad i e n t i n d i c a t e d that a s i g n i f i c a n t amount o f a polypeptide o f the size of P r 6 8 and p r e c i p i t a b l e by anti-gp69/71 i s coded f o r by v i r a l RNA of l e s s than f u l l l e n g t h s i z e . I t i s necessary t o obt a i n the t r y p t i c peptide map o f t h i s p o l y p e p t i d e to determine that i t i s a e n v gene product and not a g a g gene r e l a t e d p r o t e i n that i s a r t i f a c t u a l l y seen i n the anti-gp69/71 p r e c i p i t a t e . e n v
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Model f o r the Synthesis and P r o c e s s i n g o f R-MuLV P r o t e i n s Our r e s u l t s and those o f others (40, 41, 42, 43) i n d i c a t e that there a r e a t l e a s t two s i z e c l a s s e s of mRNAs that code f o r R-MuLV p r o t e i n s . The f u l l - l e n g t h genome s i z e 35S R-MuLV contains genes termed gag* o r core p r o t e i n s , p o l o r the DNA polymerase and e n v o r the envelope p r o t e i n s . However, only the g a g and p o l genes appear t o be t r a n s l a t e d from 35S genomic RNA (28^, 34, 39). Our f i n d i n g s i n d i c a t e that a s m a l l e r s i z e c l a s s of mRNA (-22S) codes f o r the envelope p r o t e i n s . Studies i n the a v i a n system i n d i c a t e the gene order w i t h i n the genome of non-defective ?
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In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND
GLYCOLIPIDS IN DISEASE PROCESSES
avian sarcoma v i r u s e s (another C-type RNA tumor v i r u s ) i s : 5'gag-pol-env-src -poly(A)-3 (44). Given the same gene order i n the R-MuLV genomic RNA (e.g. 'gag-pol-env'), our r e s u l t s i n d i c a t e t h a t only the f i r s t two genes are t r a n s l a t e d from v i r i o n RNA. Furthermore, our r e s u l t s suggest t h a t only one ribosome o f 15 t o 25, that has completed the t r a n s l a t i o n o f the 'gag gene, proceeds i n t o the ' p o l ' gene (26, 28, 34) ( F i g . 9 ) . This l a t t e r process we have termed read-through (28, 34) and the product o f t h i s read-through event i s a 200,000 mol. wt. p o l y p r o t e i n termed Pr200§ ° . I t contains peptide sequences and a n t i g e n i c determinants o f the 'gag' p r o t e i n and the v i r a l DNA polymerase (26, 27, 28, 34). We have shown i t to be the precursor o f the v i r i o n reverse t r a n s c r i p t a s e (26, 27). The d e t e c t i o n o f both R T - s p e c i f i c and core p r o t e i n - s p e c i f i c a n t i g e n i c determinants i n a s i n g l e precursor p o l y p r o t e i n ( P r 2 0 0 S " ) r a i s e d the f o l l o w i n g i s s u e concerning the t r a n s l a t i o n o f the v i r a l genome gene products proceeds b precursor o n l y , then t h i s would e n t a i l the s y n t h e s i s o f equimolar amounts o f g a g and ' p o l ' gene products. V i r u s p a r t i c l e s c o n t a i n much l e s s polymerase than s t r u c t u r a l p r o t e i n s 07, 8 ) . Moreover, measurement of the steady s t a t e amount o f R T - s p e c i f i c sequences i n v i r u s - i n f e c t e d c e l l s has i n d i c a t e d t h a t these sequences are present i n much s m a l l e r amounts than the v i r a l s t r u c t u r a l p r o t e i n s C7, 45). This i n d i c a t e s t h a t i f RT polypeptide sequences are i n deed made i n equimolar amounts compared t o the core p r o t e i n s , they must be q u i c k l y degraded. We have been able t o shed more l i g h t on t h i s p o i n t by comparing the amount o f RT precursors t o the amount of core p r o t e i n precursors a f t e r p u l s e - l a b e l i n g o f i n f e c t e d c e l l s (26, 27). These c o n d i t i o n s should more c l e a r l y approximate the r a t e o f s y n t h e s i s o f these p o l y p e p t i d e s . Our measurement revealed that 1/25 t o 1/10 as much R T - s p e c i f i c precursors are present i n the c e l l , a f t e r a p u l s e - l a b e l i n g , as core p r o t e i n precursors (26) . Moreover, intermediate RT precursors appear to be formed by c l e a v age of preformed Pr2008 8""P°l, w h i l e formation o f core p r o t e i n precursors i s only compatible w i t h t h i s mechanism i f very f a s t o r nascent chain cleavage o f Pr2008 S~P°l takes p l a c e , which i s not supported by our s t u d i e s w i t h amino a c i d analogs o r protease i n h i b i t o r s (26). This makes i t u n l i k e l y t h a t the pathways of synt h e s i s o f 'gag' and ' p o l ' gene products only i n v o l v e the cleavage of a common precursor. Three p o s s i b l i t i e s are t h e o r e t i c a l l y compatible w i t h our f i n d i n g s . F i r s t , the mRNA (35S i n s i z e ) coding f o r P r 2 0 0 8 S ~ P i s a minor RNA species compared t o a s m a l l e r s i z e mRNA i n v o l v e d i n the s y n t h e s i s o f 'gag' gene products. Second, there i s only one species o f mRNA coding f o r both 'gag' and ' p o l ' , but a t r a n s l a t i o n a l c o n t r o l mechanism a l l o w s the s y n t h e s i s on the mRNA of more 'gag' than ' p o l ' gene products. A t h i r d p o s s i b i l i t y i s that two c l a s s e s o f s i m i l a r s i z e mRNA (e.g. 35S) e x i s t i n the i n f e c t e d c e l l . One codes f o r 'gag' and the other codes f o r 'gag-pol'. A ,
?
T
1
ag P
a g
p O
f
T
a
a
a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
o1
10.
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ARLINGHAUS E T A L .
Figure 8. gPrQQcnv
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193
Ion exchange chromatography of tryptic digests of authentic i 68,000 mol wt polypeptide (pr68 ) made from ~ 22S mRNA from R-MuLV-infected JLS-V5 cells.
anc
t
n
cnv
e
Pr68 was labeled with S-methionine in the cell-free mRNA-dependent protein synthesizing system (as in Figure 7). gPr90 " was isolated from R-MuLV-infected JLS-V16 cells labeled with H-methionine. The proteins were purified by immunoprecipitation with anti-gp69/71 and sodium dodecyl sulfate gel electrophoresis. The bands were digested with TPCK-trypsin and fractionated on a cation exchange column (30). env
35
c
v
3
Translotionol control
t
35SI mole P r 8 0 Pr65
Pol
Q>
Poly (A) -vw\
T
0'
gPr90
fl0g
H—-1
20S ,nv
viral mRNA
— I mole
-
,afl
4
\
Pr 27
Pr 42
4
4 \
pi5
\
gp 69/71
iZT gag-pol
\
pl2E
pl2 p30 plO
1/8 - 1/15 mole Pr 200
pl5E
1 pol
- Pr 145
I = initiation T = termination
•
Polymerase
Figure 9.
A model illustrating the overall steps involved in the synthesis and processing of Rauscher murine leukemia viral proteins
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
194
GLYCOLIPIDS IN DISEASE PROCESSES
c o n s i d e r a t i o n o f other a v a i l a b l e i n f o r m a t i o n favors the second o r t h i r d p o s s i b i l i t y . Thus, the major species o f v i r a l mRNA i n murine and a v i a n RNA tumor v i r u s - i n f e c t e d c e l l s has a s i z e o f 35S, but species o f 21S and 16S have a l s o been detected (40, 41, ^ 2 , 43). Moreover, f u r t h e r s t u d i e s i n d i c a t e that the 35S RNA i s the main species present i n polyribosomes p r e c i p i t a b l e w i t h anti-p30 mono-specific serum (46). Studies i n a v i a n systems a l s o suggest that the f u l l - l e n g t h genomic RNA i s the mRNA f o r 'gag and ' p o l ' gene products (38, 47). These observations make i t u n l i k e l y that there i s a s m a l l s i z e mRNA (-16S) on which the m a j o r i t y o f the 'gag gene products are synthesized and d i s f a v o r the p o s s i b i l i t y that a l a r g e s i z e species (-35S) o f v i r a l mRNA, such as one able to code f o r P r 2 0 0 , c o n s t i t u t e a minor p o p u l a t i o n . I t i s d i f f i c u l t t o e l i m i n a t e the p o s s i b i l i t y that there are indeed two c l a s s e s o f 35S o r f u l l - l e n g t h genomic s i z e RNAs, a major one coding f o r 'gag' gene products and a minor one coding f o r 'gag' and ' p o l ' gen that s t o r e d 35S v i r a l RN gene a t the expense o f the 'gag' gene precursors than f r e s h batches 35S v i r a l RNA (Murphy and A r l i n g h a u s , unpublished r e s u l t s ) . This r e s u l t i s c o n s i s t e n t w i t h the idea that some s t r u c t u r a l a l t e r a t i o n ^ ) of 35S RNA genomic i s r e s p o n s i b l e f o r P r 2 0 0 g g ~ P s y n t h e s i s . This a l t e r a t i o n could be brought i n t o p l a y by i n t e r a c t i o n o f the 35S v i r a l RNA w i t h a second molecule. A candidate f o r t h i s second molecule would be a RNA s i n c e pronease-treated v i r a l 35S RNA does not a l t e r the frequency o f P r 2 0 0 ~ P synt h e s i s over non-treated RNA (Murphy and A r l i n g h a u s , unpublished results). Our r e s u l t s i n d i c a t e t h a t the envelope gene i s probably never t r a n s l a t e d from f u l l length genomic s i z e RNA but that a -22S i n t r a c e l l u l a r mRNA serves as a mRNA f o r the envelope p r o t e i n . The p o l a r i t y o f t h i s mRNA i s unknown but a s i m i l a r v i r a l envelope mRNA from a v i a n sarcoma v i r u s i n f e c t e d c e l l s has the same p o l a r i ty as v i r a l genomic RNA (47, 48, 49). Therefore, we would assume that -22S mRNA composed o f sequences from the 3' o n e - t h i r d o f R-MuLV genomic RNA i s the mRNA f o r the envelope p r o t e i n s . We would a l s o assume that t h i s RNA i s capped and contains some unt r a n s l a t e d sequences found a t the 5' end o f the 35S genomic RNA (48). 1
1
g a g p
o
a
g a g
o1
o 1
Model f o r the Synthesis and P r o c e s s i n g o f V i r a l Envelope P r o t e i n s As the model i n F i g . 10 shows, the i n i t i a l t r a n s l a t i o n product of the -22S 'env' mRNA i s a 68,000 mol. wt. non-glycosylated polypeptide that contains peptide sequences o f the major g l y c o p r o t e i n (gp69/71) and non-glycosylated p l 5 ( E ) . The next step i s shown to be the g l y c o s y l a t i o n o f the p o l y p r o t e i n producing g P r 9 0 . A l t e r n a t e l y , i t i s possible that inside infected c e l l s g l y c o s y l a t i o n occurs p r i o r t o formation o f the complete 'env' gene product which would r e s u l t i n the immediate formation o f e n v
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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e n v
g P r 9 0 . I f t h i s were the case, P r 6 8 would only be seen under c o n d i t i o n s o f c e l l - f r e e t r a n s l a t i o n and not i n whole c e l l s . However, i t has not been e s t a b l i s h e d whether or not some P r 6 8 i s formed i n i n f e c t e d c e l l s . Cleavage and f u r t h e r g l y c o s y l a t i o n (fucose residues are added) of g P r 9 0 v y i e l d s gp69/71 and pl5(E) which probably e x i s t s as a complex held together by d i s u l f i d e bonds and p o s s i b l y a d d i t i o n a l non-covalent bonds. The b a s i c u n i t of the p r o t r u d i n g s p i k e - g l y c o p r o t e i n knob i s b e l i e v e d to be t h i s gp69/71-pl5(E) complex. The a s s o c i a t i o n of such b a s i c u n i t s would then r e s u l t i n formation of the spike-knob s t r u c t u r e that t r a v e r s e s and p r o j e c t s through the l i p i d envelope of the v i r u s ( F i g . 11). The r o l e of v i r a l p r o t e i n pl2(E) i n the s t r u c t u r e of the v i r i o n i s unknown. Since, i t does not appear to form a complex w i t h gp69/71 (50), we assume that i t may be an a r t i f a c t of aged v i r u s r e s u l t i n g from the p r o t e o l y s i s o f the spike-knob s t r u c t u r e which i n t u r n leads to a s m a l l p o r t i o n of the e n v
en
V i r u s Assembly Cleavage of v i r a l p o l y p r o t e i n s c o n s t i t u t e s a predominant f e a t u r e i n the assembly of many animal and b a c t e r i a l v i r u s e s ( f o r reviews see 51, J52, J53, 54) . RNA tumor v i r u s e s are no exception. We have shown that actinomycin D reduced the r a t e of cleavage of Pr65gag (55) and have suggested that the r a t e o f cleavage o f Pr65S £ to the mature v i r a l - p r o t e i n s i s c a t a l y z e d by the v i r a l genomic RNA. Recent experiments have a l s o shown that the c l e a v age r a t e of Pr200g "~ i s a l s o a f f e c t e d by i n h i b i t i n g v i r a l RNA s y n t h e s i s (Kopchick and A r l i n g h a u s , unpublished r e s u l t s ) . We note that the cleavage of Pr80S S to P r 6 5 g i s not a f f e c t e d under these c o n d i t i o n s nor i s the cleavage of g P r 9 0 . However, cleavages of Pr65gag and P r 2 0 0 " " P do occur (but at a slower r a t e ) i n the absence of v i r a l RNA and, i n t e r e s t i n g l y , the reverse t r a n s c r i p t a s e can be incorporated i n t o v i r i o n s that are d e f i c i e n t of v i r a l RNA (56). Based on a number of s t u d i e s from my own labor a t o r y (55), we have proposed a model f o r the assembly of R-MuLV p a r t i c l e s ( F i g . 11). In t h i s model, P r 6 5 g g r a t h e r than the mature v i r a l p r o t e i n s c o n s t i t u t e the i n i t i a l assembly u n i t . U n i t s of P r 6 5 g g assemble, presumably at the c e l l membrane, i n t o a s t r u c t u r e that remains open on the i n s i d e , due to the e l e c t r o s t a t i c r e p u l s i o n of the b a s i c p o r t i o n (plO) of P r 6 5 g g , which i s then cleaved to form the v i r a l p r o t e i n s i n c l u d i n g plO. V i r a l plO i n t u r n i s found i n the v i r i o n i n a s s o c i a t i o n w i t h the v i r a l RNA (5, 57). This model a l s o proposes that P r 2 0 0 g ~ i s incorpor a t e d i n t o the v i r i o n by means of i t s s i m i l a r i t y i n s t r u c t u r e t o P r 6 5 g . In f a c t , P r 2 0 0 g g " * contains a l l the a n t i g e n i c d e t e r minants and peptide sequences that are found i n P r 6 5 g g plus those of the reverse t r a n s c r i p t a s e . Thus, Pr200g g~P°l would s u b s t i t u t e f o r s m a l l number (30-50) o f the P r 6 5 molecules r e s u l t i n g a
ag
p
a
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a
a
a
g a
a g
a
p o 1
po1
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a
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In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
196
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES gp69/71
20-22S
mRNA
-p15(E) -...poly (A)
Cap.
3'
Translation
Pr68
,
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s Translation and / Glycosylation
Glycosylation
gPr90
LTTl
erv
|
V
f
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COOH
Carbohydrate Chains Cleavage and Fucosylation
gp69/71 -p15(E) Complex
gp69/71
p15(E)
s-s
Viral Spike and Knob Assembly Figure 10.
Synthesis of Rauscher murine leukemia virus envelope proteins
9a9 f>0
^-Pr200 - ' vRNA
-Unc leaved Pr65W
| Cleavage of Pr65W and Pr200W-i>
01
Mature core proteins Budding virus
Cell membrane Figure 11. A model showing the assembly and budding of Rauscher murine leukemia virus particles
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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i n the i n c l u s i o n o f RT i n the v i r i o n . _ A s u b s t a n t i a l amount o f cleavage o f P r 6 5 fend P r 2 0 0 ° ) i s proposed i n the model i n order to a l l o w v i r u s r e l e a s e . V i r a l RNA, due t o charge n e u t r a l i z a t i o n , c a t a l y z e s the cleavage o f Pr65§ and P r 2 0 0 " . T h i s r e s u l t s i n a more r a p i d r a t e o f assembly of RNA-containing v i r a l c o r e s , and, i f p r e c u r s o r c l e a v age i s viewed to p l a y a p a r t i n the budding process, i n a f a s t e r r a t e o f r e l e a s e o f RNA-containing v i r u s p a r t i c l e s . We f u r t h e r propose that host c e l l RNA can s u b s t i t u t e t o a c e r t a i n degree f o r v i r a l RNA i n c a t a l y z i n g the cleavages of P r 6 5 and P r 2 0 0 " P , but host c e l l u l a r RNA i s packaged to a much l e s s e r extent than v i r a l RNA s i n c e v i r u s i s o l a t e d a f t e r actinomycin D treatment exh i b i t e d a lower r a t i o o f RNA to p r o t e i n than c o n t r o l p r e p a r a t i o n s of v i r u s (55). T h i s suggests the e x i s t a n c e of s p e c i f i c r e c o g n i t i o n by the assembled v i r a l s t r u c t u r e s o f v i r a l genomic RNA. Whether the b a s i s f o r t h i s r e c o g n i t i o n l i e s i n the s p e c i f i c i n t e r a c t i o n between some o f the v i r a v i r a l RNA (6) remains t In the above mentioned model of assembly, we have p o s t u l a t e d t h a t cleavages o f P r 6 5 and P r 2 0 0 8 ~ P i s probably r e q u i r e d f o r v i r u s budding and r e l e a s e . There i s a p o s s i b i l i t y , however, that Pr658 8 and P r 2 0 0 ~ may be cleaved not d u r i n g budding but a f t e r r e l e a s e o f the v i r u s from the c e l l . For many y e a r s , C-type p a r t i c l e s have been observed to undergo a p o s t - r e l e a s e maturation process i n which the core center of the v i r i o n c o l lapses i n t o a condensed s t r u c t u r e that i s c l e a r l y d i f f e r e n t from the organized c o n c e n t r i c c o i l s observed i n the core of budding p a r t i c l e s (58, 59, 60). The s i g n i f i c a n c e o f t h i s maturation process remains u n c e r t a i n . The recent r e s u l t s of Yoshinaka and L u f t e g (61) suggest that there i s a p o s i t i v e c o r r e l a t i o n between the amount of P r 6 5 8 8 i n v i r a l cores and the amount o f immature cores. However, i t remains to be shown whether o r not the c l e a v age o f Pr658 8 can occur i n r e l e a s e d v i r i o n s and i f so that the changes i n r e l e a s e d v i r i o n s (immature to mature) a r e r e l a t e d t o precursor cleavage. A number o f _ l i n e s evidence suggest that cleavage o f P r 6 5 8 8 and P r 2 0 0 ° occur mainly p r i o r t o v i r a l r e l e a s e (62) and p r i o r t o the immature t o mature v i r u s t r a n s i t i o n that takes p l a c e i n the c u l t u r e f l u i d . 1
g a g
ag
g a g
P
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g a g
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Literature Cited 1. Pinter, A. and Fleissner, E. Virology (1977) 83, 417. 2. Leamnson, R. N., Shander, M. H. M. and Halpern, M. S. Virology (1977) 76, 437. 3. Witte, O. N., Tsukamoto-Adey, A. and Weissman, I . L. Virology (1977) 76, 539. 4. Bolognesi, D. P . , Montelaro, R. C., Frank, H. and Schafer, W. Science (1978) 199, 183. 5. Davis, J., Scherrer, M., Tsai, W. P . , and Long, C. J . Virol. (1976) 18, 709.
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32. Butterworth, B. E . , and Rueckert, R. R. J . Virol. (1972) 9, 823. 33. Naso, R. B . , Arcement, L. J., Wood, T. G., Saunders, T. E . , and Arlinghaus, R. B. Biochim. Biophys. Acta (1975) 383, 195. 34. Murphy, J r . , E. C., and Arlinghaus, R. B. Virology (1978) In press. 35. Kerr, I. M., Olshevsky, U . , Lodish, H . , and Baltimore, D. J. Virol. (1975) 18, 627. 36. Pawson, T., Martin, G. S., and Smith, A. E. J . Virol. (1976) 19, 950. 37. Von Den Helm, K . , and Duesberg, P. H. Proc. Natl. Acad. Sci. (1975) 72, 614. 38. Paterson, B. M., Marciani, D. J., and Papas, T. S. Proc. Natl. Acad. Sci. USA (1977) 74, 4951. 39. Philipson, L., Andersson, P., Olshevsky, U . , Weinberg, R., and Baltimore, D. Cell (1978) 13 189 40. Fan, H. and Baltimore 41. Gielkens, A. L. J., Bloemendal, H. Proc. Natl. Acad. Sci. USA (1976) 73, 356. 42. Shanmugam, G., Bhadure, S. and Green, M. Biochim. Biophys. Res. Commun. (1974) 56, 697. 43. Shincarial, A. and Joklik, W. Virology (1973) 56, 532. 44. Duesberg, P. H . , Wang, L. H . , Mellan, P . , Mason, W. S., and Vogt, P. K. "ICN-UCLA Symposia on Molecular and Cellular Biology" Vol. IV, pp 107. Edited by D. Baltimore, A. S. Huang, and C. F. Fox. Academic Press, Inc., New York, New York. 1976. 45. Chen, J . H. and Hanafusa, H. J . Virol. (1974) 13, 340. 46. Mueller-Lantsch, N. and Fan, H. Cell (1976) 9, 579. 47. Haywood, W. S. J . Virol. (1977) 24, 47. 48. Mellon, P. and Duesberg, P. H. Nature (1977) 270, 63. 49. Stacey, D. W., Allfrey, V. G., and Hanafusa, H. Proc. Natl. Acad. Sci. USA (1977) 270, 631. 50. Montelaro, R. C., Sullivan, S. J., and Bolognesi, D. P. Virology (1978) In press. 51. Casjens, S. and King, J . Annu. Rev. Biochem. (1975) 44, 555. 52. Hershko, A. and Fry, M. Annu. Rev. Biochem. (1975) 44, 775. 53. Korant, B. D. "Cold Spring Harbor Symposium on Control of Cell Proliferation" Vol. 21, p. 621. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1975. 54. Showe, M. H. and Kellenberg, E. "Control Processes in Virus Multiplication" p. 407. Cambridge University Press, Cambridge, England. 1975. 55. Jamjoom, G. A . , Naso, R. B . , and Arlinghaus, R. B. J . Virol. (1976) 19, 1054. 56. Levin, J . G., Grimley, P. M., Ramseur, J . M., and Berezesky, I. K. J . Virol. (1974) 14, 152. 57. Fleissner, E. and Tress, E. J . Virol. (1973) 12, 1612. 58. Fleissner, E . , Ikeda, H . , Tung, J . S., Vitetta, E . , Tress, E., Hard, J r . , W., Stockert, E . , Boyse, E. A . , Pincus, T., and
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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59. Dalton, A. J. and Haguenau, F. "Ultrastructure of Animal Viruses and Bacteriophages" pp. 255. Eds. A. J. Dalton and F. Haguenau. Academic Press, New York, New York. 1973. 60. deHarven, E. Adv. Virus Res. (1974) 19, 221. 61. Yoshinaka, Y. and Luftig, R. B. Proc. Natl. Acad. Sci. USA (1977) 74, 3445.
62. Jamjoom, G. A . , Ng, V. L., and Arlinghaus, R. B. J . Virol. (1978) 25, 408. RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11 Glycoprotein Inhibitor of Lipoprotein Lipase from Aortic Intima—Its Possible Role in Atherosclerosis PREMANAND V. WAGH Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, AR 72206
Early observations th f glycoprotein i th mammalian arterial wal -Spreer et a l , 1960; Berenso , 1962) Althoug siderable research interest is centered upon the relationship of glycoproteins in the aortic wall to atherosclerosis and ageing, little is known in regard to the structure and function of these macromolecules. Several soluble glycoproteins have been purified from bovine aorta after extraction of the tissue with neutral buffers (Radhakrishnamurthy et a l , 1964, Maier and Buddecke, 1971). The concentration of arterial glycoproteins in atherosclerosis and in ageing is a subject of question and is being actively investigated. The wet weight of the aorta has been found to be increased in atherosclerosis and among the aged (Manley and Mullinger, 1967). Our data (Wagh et a l , 1973), shown in Table I, provide evidence that the glycoprotein concentration increased significantly in atherosclerotic tissue as expressed by an increase in total neutral sugars, hexosamine and sialic acid. These results suggest that the increased glycoprotein concentration may have a functional role in the formation of atherosclerotic plaque. Several functions have been ascribed to arterial glycoproteins such as transplantation rejection and maintenance of structural integrity (Anderson, 1976). Furthermore, Ishii (1971) demonstrated that preparations of crude glycoprotein obtained from various organs including the aorta of dog inhibited dextran sulfate released lipoprotein lipase (LPL) activity of human plasma. LPL catalyzes the hydrolysis of the triglyceride moiety of chylomicrons and very low density lipoproteins (Eisenberg and Levi, 1975), and is normally located on the surface of endothelial cells where it is physiologically active in the normal clearance of lipoprotein-bound triglycerides (Robinson, 1970). Since the surface of the intimal layer of aortic wall is susceptive to continuous hemodynamic insult and thus may be i n volved in the genesis of atherosclerosis and other physiological events, we focussed our attention here for the purpose of isolation of glycoprotein. One of the shortcomings in the isolation 201 This chapter not subject to U . S . copyright Published 1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
of p u r i f i e d g l y c o p r o t e i n from the i n t i m a l region has been to separate i t from the r e s t of the a o r t i c w a l l . A new procedure (dermatome procedure) was developed i n our l a b o r a t o r y (Roberts et a l , 1974) that r a p i d l y and r e l i a b l y separates the intima i n s u f f i c i e n t q u a n t i t i e s from both porcine and human a o r t a . Studies on Porcine I n t i m a l G l y c o p r o t e i n ( L i p o l i p i n ) . We r e ported on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a novel g l y c o p r o t e i n from the i n t i m a l r e g i o n of porcine aorta (Wagh and Roberts 1972). Swine a o r t a was chosen because i n t h i s s p e c i e s , the aorta i s r e l a t i v e l y f r e e from a t h e r o s c l e r o t i c l e s i o n s and the m a t e r i a l i s r e a d i l y a v a i l a b l e . The p u r i f i c a t i o n procedure involved e x t r a c t i o n of i n t i m a l t i s s u e with n e u t r a l b u f f e r , (Nlfy^SO^ p r e c i p i t a t i o n between 40-90% s a t u r a t i o n and two DEAE-cellulose chromatographic steps. The p u r i f i e d g l y c o p r o t e i n was homogeneous upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n (4.86 S) and by polyacrylamide disc-gel electrophoresis p r o t e i n was obtained fro Due to the presence of equimolar glucose and galactose and the absence of hydroxylysine i n the molecule, i t was suggested that t h i s g l y c o p r o t e i n was unique i n i t s c h a r a c t e r i s t i c s and t h e r e f o r e of a new type. The i s o e l e c t r i c p o i n t of the p u r i f i e d glycoprot e i n was 4.3 as observed by a n a l y t i c a l i s o e l e c t r i c focusing (Baig and Ayoub, 1976). Subsequent s t u d i e s (Roberts and Wagh, 1976) revealed that sodium dodecyl s u l f a t e - polyacrylamide g e l e l e c t r o p h o r e s i s of the n a t i v e and i t s S-carboxyamidomethyl d e r i v a t i v e a t d i f f e r e n t polyacrylamide concentrations d i d not a f f e c t the molecular weight (72,000 daltons) i n d i c a t i n g the absence of subunits. The carboxyt e r m i n a l amino a c i d was found to be s e r i n e . Attempts to determine the i d e n t i t y of the amino a c i d i n d i c a t e d that the amino group was not f r e e . The g l y c o p r o t e i n d i d not contain an a l k a l i - l a b i l e (0g l y c o s i d i c ) carbohydrate-peptide linkage as tested by $-eliminat i o n r e a c t i o n . The r e l e a s e of monosaccharides from the i n t a c t g l y c o p r o t e i n as a f u n c t i o n of time was studied employing m i l d a c i d h y d r o l y s i s (0.5 M HC1, 80°C) and a l s o by the use of neuraminidase, a-D- and $-D-glucosidases and $-D-N-acetylglucosaminidase. From the observations on the r e l e a s e of monosaccharides and analogy with standard features determined by other i n v e s t i g a t o r s on s o l u b l e a o r t i c g l y c o p r o t e i n s (Radhakrishnamurthy e t a l , 1964; Radhakushnamurthy and Berenson, 1966; Klemer and Nager, 1967; Maier and Buddecke, 1971), a p r e d i c t i o n was made as to the general features of the carbohydrate moiety of the g l y c o p r o t e i n ( F i g . 1 ) . This p o s t u l a t e d s t r u c t u r e must s t i l l withstand r e s u l t s of f u t u r e i n v e s t i g a t i o n s i n c l u d i n g methylation s t u d i e s , o x i d a t i o n with p e r i o date and the use of other s p e c i f i c g l y c o s i d a s e s . The p u r i f i e d porcine i n t i m a l g l y c o p r o t e i n was tested f o r i t s LPL i n h i b i t o r y a c t i v i t y by employing post-heparin dog plasma as the source of enzyme and E d i o l ( s t a b i l i z e d coconut o i l emulsion) as the t r i g l y c e r i d e s u b s t r a t e . Because i t i n h i b i t e d post-heparin
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
203
Lipase
TABLE I C o n c e n t r a t i o n of Component Sugars of Normal and A t h e r o s c l e r o t i c Aortae Component
Sclerotic
Normal
2 Wet weight Xmg/cm ) DDAF (mg/cm ) T o t a l carbohydrate (mg/cm ) Hexose (ug/cm ) Hexosamine (ug/cm ) Uronic a c i d (ug/cm^) S i a l i c a c i d (ug/cm^) T o t a l carbohydrate (ug/m Hexose (ug/mg DDAF) Hexosamine (ug/mg DDAF) Uronic a c i d (ug/mg DDAF) S i a l i c a c i d (ug/mg DDAF)
250.4 (a) 36.9 (b) 2.30 (a) 978 (a) 698 (c) 308 (NS) 317 (b)
195.7 39.3 2.10 870 651 297 285
2
(a) 19.4 (a) 8.5 (NS) 8.8 (a)
17.0 7.9 7.5
The c o n c e n t r a t i o n o f components are expressed as weight per u n i t area and weight per u n i t dry, d e f a t t e d , a s h - f r e e r e s i d u e (DDAF). A l l v a l u e s represent averages from 15 samples of aortae obtained from i n d i v i d u a l s of 7 0 . 3 + 2 . 8 (mean + S.E.M.) years of age. P a i r e d t t e s t : (a) = P<0.01; (b) = P<0.05; (c) = P<0.02; (NS) = not s i g n i f i c a n t . From Wagh e t a l (1973), w i t h p e r m i s s i o n from E l s e v i e r / N o r t h - H o l l a n d Biomedical P r e s s .
NeuAc
Glu
Gal
Gal
Glu
GlcNAc
GlcNAc
GlcNAc
y
x ^
Maojf
(Fuc)?
I
i
^jyian
Glu
GlcNA^c
Gal Biochimica et Biophysica Acta AspNH (?) Figure 1. A predicted structure for the carbohydrate moiety of porcine intimal glycoprotein. NeuAc, N acetyl-neuraminic acid; GlcNAc, N-acetyl-D-glucosamine. (From Roberts and Wagh [1976]) 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
plasma LPL, the molecule was named " l i p o l i p i n " (from l i p o p r o t e i n l i p a s e i n h i b i t o r ) (Wagh, 1975). The i n h i b i t i o n of LPL a c t i v i t y at v a r i o u s l i p o l i p i n concent r a t i o n s and a t r i g l y c e r i d e c o n c e n t r a t i o n of 2.5 mM i s shown i n F i g . 2. I n h i b i t i o n of LPL increased e x p o n e n t i a l l y with l i p o l i p i n c o n c e n t r a t i o n . At the t r i g l y c e r i d e concentration used, the r e a c t i o n v e l o c i t y was reduced 50% when the concentration of l i p o l i p i n was 1.5 x 10~5 M. To determine the nature of i n h i b i t i o n , we measured the v e l o c i t y of l i p o l y s i s at v a r i o u s concentrations of t r i g l y c e r i d e and the i n h i b i t o r . The i n h i b i t i o n was observed to be non-competitive ( F i g . 3). Further s t u d i e s (unpublished data) i n our l a b o r a t o r y i n d i c a t e d that porcine l i p o l i p i n i n h i b i t s p u r i f i e d bovine m i l k LPL (Egelrud and O l i v e c r o n a , 1972) when r a d i o a c t i v e t r i g l y c e r i d e emulsion s t a b i l i z e d by gum a r a b i c ( H e r n e l l et a l , 1975) was used as the s u b s t r a t e . However, the mechanism by which l i p o l i p i stood due to complex natur The s t a b i l i t y of l i p o l i p i n under v a r i o u s c o n d i t i o n s was examined by e l e c t r o p h o r e s i s on polyacrylamide g e l . L i p o l i p i n i s unstable a f t e r approximately 2 weeks when stored i n n e u t r a l Tris« HC1 b u f f e r or as a f r e e z e - d r i e d powder. The i n s t a b i l i t y i s i n d i cated by a d d i t i o n a l bands i n the g e l and the l o s s of i t s i n h i b i t o r y property (Wagh, 1975). Studies on Human I n t i m a l G l y c o p r o t e i n . We extended our i n v e s t i g a t i o n of LPL i n h i b i t o r to human a t h e r o s c l e r o t i c intima (Wagh and O l i v e c r o n a , 1978). A t h e r o s c l e r o t i c human aortae (Grade III-IV) were obtained at necropsy from male veterans 60-80 years of age. I n t i m a l l a y e r was separated by the "dermatome procedure" and intima powder was prepared as described p r e v i o u s l y (Roberts et a l , 1974). The y i e l d of intima powder was 1.5 - 2.0 g per 100 g of wet weight of aortae. P u r i f i c a t i o n of human LPL i n h i b i t o r demanded that the o r i g i n a l procedure f o r porcine t i s s u e (Wagh and Roberts, 1972) be modi f i e d (Wagh and O l i v e c r o n a , 1978). B r i e f l y , intima powder (5 g) was e x t r a c t e d twice with 20 v o l of acetone at 4°C f o r 30 min. The acetone powder was d r i e d at 25°C f o r 30 min under vacuum. This step r e s u l t e d i n the removal of 0.75 g l i p i d s and was found to be necessary because the presence of these l i p i d s i n t e r f e r e d i n subsequent e x t r a c t i o n procedures. The acetone powder was ext r a c t e d two times each f o r a p e r i o d of 24 h with 20 v o l of e x t r a c t i o n b u f f e r (0.05 M Tris-HHCl - 1 mM EDTA - 0.3 M NaCl, pH 7.4). The ammonium s u l f a t e p r e c i p i t a t e of the e x t r a c t between 40-80% s a t u r a t i o n was c o l l e c t e d , d i s s o l v e d i n 5 mM T r i s * H C l cont a i n i n g 1 mM EDTA and 50 mM NaCl, pH 7.4 b u f f e r and d i a l y z e d exh a u s t i v e l y against the same b u f f e r . The d i a l y z e d s o l u t i o n was designated as the ammonium s u l f a t e f r a c t i o n . T h i s f r a c t i o n upon chromatography on a DEAE-cellulose column r e s u l t e d i n three peaks which contained m a t e r i a l that i n h i b i t e d LPL a c t i v i t y ( F i g . 4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
Lipase
Advances in Experimental Medicine and Biology
Figure 2. Inhibition of LPL as a function of lipolipin concentration. Incubation mixture contained 0.2 mL of postheparin plasma, 0.5 mL of buffer (0.2M NH,,OH-NH Cl at a pH of 8.6) containing various amounts of lipolipin and 0.1 mL of Ediol (20 micromoles triglyceride per milliliter). Final concentration of triglyceride was 2.5mM. Percent inhibition was calculated as the decrease in LPL activity as related to assay mixtures without added lipolipin. (From Wagh [1975]) h
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
180-
Advances in Experimental Medicine and Biology Figure 3. Dixon plot of LPL activity. Incubation mixture contained 0.2 mL of post-heparin plasma, 0.5 mL of buffer (0.2M NH, OH-NH,,Cl at a pH of 8.6 containing various amounts of triglycerides). Velocity is expressed as micromoles of free fatty acids liberated per milliliter of post-heparin plasma per minute. Final concentrations of triglyceride were: (•—0.7mM; (A—A; 1.6mM; (X—X) 2.5mM; and (O—O) 4.3mM. (From Wagh [1975]) t
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of
Lipoprotein
Lipase
207
Peak 1 contained l a r g e amounts of hemoglobin which s e r i o u s l y i n t e r f e r r e d with the LPL assay. The p r o t e i n m a t e r i a l i n peak 2 e l u t e d at the same i o n i c strength of the gradient as that f o r porcine l i p o l i p i n . I t s homogeneity was assessed by polyacrylamide g e l e l e c t r o p h o r e s i s , a n a l y t i c a l i s o e l e c t r i c focusing and sediment a t i o n v e l o c i t y measurement. E l e c t r o p h o r e t i c a n a l y s i s as observed by scanning the g e l stained with Coomassie B r i l l i a n t Blue dye at 520 nm revealed one major and three minor components. The major component c o n s t i t u t e d approximately 80% of the t o t a l p r o t e i n . The e l e c t r o p h o r e t i c m o b i l i t y of the major component a f t e r s t a i n i n g f o r both the p r o t e i n and carbohydrate ( p e r i o d i c a c i d - S c h i f f s t a i n ) was s i m i l a r to that of h i g h l y p u r i f i e d porcine l i p o l i p i n . A n a l y t i c a l i s o e l e c t r i c f o c u s i n g i n a pH 4-6 ampholyte system r e s u l t e d i n a p a t t e r n of one major and two minor p r o t e i n bands as v i s u a l i z e d by Coomassie Blue s t a i n i n g . The major component e l e c t r o f o c u s e d at pH 4.3. Upon u l t r a c e n t r i f u g a t i o n major species which sedimente content of the peak 2 m a t e r i a l was 2.55%. The carbohydrate contained hexosamine, glucose, galactose, mannose, fucose and s i a l i c a c i d i n a molar r a t i o of 3:1:2:2:1:1, r e s p e c t i v e l y with s i a l i c a c i d taken as u n i t y . This m a t e r i a l e l u t e d from Bio-Gel P-150 i n the same p o s i t i o n as described f o r porcine l i p o l i p i n (Roberts and Wagh, 1976). A p a r t i a l l y p u r i f i e d p r e p a r a t i o n was employed f o r the k i n e t i c s t u d i e s . Instead of chromatography on DEAE-cellulose column, a batch-wise DEAE-cellulose f r a c t i o n a t i o n method was used (Wagh and O l i v e c r o n a , 1978). The p r e p a r a t i o n contained a l l the p r o t e i n i n peak 2 with some overlapping p r o t e i n s contributed from peak 3 (approximately 20%) but was f r e e from p r o t e i n s i n peak 1 ( F i g . 4). This m a t e r i a l i n h i b i t e d the a c t i v i t y of milk LPL against longchain t r i g l y c e r i d e s under a l l c o n d i t i o n s that we have studied (Wagh and O l i v e c r o n a , 1978). The r e l e a s e of f r e e f a t t y a c i d s was l i n e a r with time both with and without i n h i b i t o r , i n d i c a t i n g that the i n h i b i t o r d i d not cause a p r o g r e s s i v e i n a c t i v a t i o n of the enzyme with time. I n h i b i t i o n of both the b a s a l a c t i v i t y and the serum-stimulated a c t i v i t y was the same f o r upto a 7-fold increase of enzyme concentration. I t was observed that the i n h i b i t i o n was non-competitive with respect to serum ( F i g . 5). When the amount of t r i g l y c e r i d e substrate was increased at a constant l e v e l of serum, a c t i v i t y i n the absence of i n h i b i t o r f i r s t increased but then decreased at high concentrations of t r i g l y c e r i d e ; however, the i n h i b i t i o n was r e l i e v e d at high l e v e l s of t r i g l y c e r i d e subs t r a t e ( F i g . 6). Therefore, the i n h i b i t i o n may be competitive with respect to t r i g l y c e r i d e s u b s t r a t e . Concluding Remarks: I should l i k e to end by posing four questions to which we p r e s e n t l y do not have answers or our knowledge i s at best incomplete. F i r s t , are porcine and human i n t i m a l l i p o l i p i n s s i m i l a r ? There i s some evidence that both of these molecules share c e r t a i n c h a r a c t e r i s t i c s i n common, e.g., molecular
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Atherosclerosis Figure 4. Chromatography of ammonium sulfate fraction on DEAE-cellulose. The ammonium sulfate fraction was chromatographed on a DEAE-cellulose column. The elution was initiated with 5mM Tris • HCl - ImM EDTA-0.05M NaCl, pH = 7.4 (standard buffer). The elution was initiated with 450 mL of standard buffer and 0.25M NaCl in the same buffer. Fractions of 6 mL were collected. The flow rate was 53 mL/hr. (From Wagh and Olivecrona [1978])
0
10
20 30 40 50 60 SERUM (/il/ml assay mixture)
70
80
Atherosclerosis Figure 5. The effect of various concentrations of human serum on LPL inhibition. (O—O) control; (•—•) 356 fig inhibitor per mL assay. For details see Kef. Wagh and Olivecrona (1978).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
0
0-5
Inhibitor
1-0
of Lipoprotein
1-5
0 L I V E 01L
2-0
Lipase
2-5
209
3-0
6-0
(mg/ml assay mixture)
Atherosclerosis
Figure 6. inhibition.
The effect of various concentrations of olive oil on LPL (O—O) control; (•—•) 625 fig inhibitor per mL assay. For details see Kef. Wagh and Olivecrona (1978).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOLIPIDS IN
DISEASE PROCESSES
weight, e l e c t r o p h o r e t i c m o b i l i t y and i s o e l e c t r i c p o i n t . Although the s t r u c t u r e of the carbohydrate moiety f o r the porcine molecule has been p r e d i c t e d (Roberts and Wagh, 1976) the p r e c i s e sequence of sugars remains to be confirmed and compared w i t h that of the human molecule. The second question i s - what i s the mechanism by which l i p o l i p i n i n h i b i t s LPL a c t i v i t y i n v i t r o ? We have p o s t u l a t e d from k i n e t i c s t u d i e s that the i n h i b i t i o n of LPL may be at l e a s t p a r t l y due to covering of the l i p i d - w a t e r interphase by the i n h i b i t o r thereby denying the enzyme access to the l i p i d s u b s t r a t e (Wagh and O l i v e c r o n a , 1978). Although t h i s has been suggested as a mechanism f o r the i n h i b i t i o n of p a n c r e a t i c l i p a s e by bovine serum albumin (Brockerhoff, 1971), f u r t h e r s t u d i e s are needed to confirm our hypothesis. T h i r d , which c e l l s i n the i n t i m a synthesize l i p o l i p i n ? I t has been demonstrated tha glycoprotein similar t 1976). Further immunological s t u d i e s on the c r o s s - r e a c t i o n between a n t i - l i p o l i p i n against i n t i m a l l i p o l i p i n and v a l v u l a r g l y c o p r o t e i n i n d i c a t e d that the p r e c i p i t i n bands formed due to c r o s s - r e a c t i o n fused w i t h each other (unpublished d a t a ) . I t seems t h e r e f o r e , that l i p o l i p i n i s present i n the mesenchymal t i s s u e s . Further h i s t o l o g i c a l and immunological s t u d i e s should provide i n f o r m a t i o n as to the l o c a l i z a t i o n of l i p o l i p i n i n the v a s c u l a r tissue. F i n a l l y , we come to the question of the f u n c t i o n of LPL i n h i b i t o r i n v i v o . Large blood v e s s e l s such as bovine t h o r a c i c a o r t a c o n t a i n LPL (Henson and Schotz, 1975; D i c o r l e t o and Z i l v e r smit, 1975). Although the c o n c e n t r a t i o n of LPL i n h i b i t o r i n the normal and diseased s t a t e s i s not known, i t i s p o s s i b l e that i n creased s y n t h e s i s of the i n h i b i t o r during atherogenesis may r e s u l t i n decreased LPL a c t i v i t y thereby a f f e c t i n g the normal l i p o l y t i c process. I f the i n h i b i t o r i s a major c o n s t i t u e n t of t o t a l g l y c o p r o t e i n content i n a t h e r o s c l e r o t i c a o r t a , the higher c o n c e n t r a t i o n of g l y c o p r o t e i n s i n a t h e r o s c l e r o t i c plaques as compared to that i n the uninvolved regions of the a o r t a (Wagh et a l , 1973) may be a r e f l e c t i o n of an increased c o n c e n t r a t i o n of the i n h i b i t o r i n diseased s t a t e . This s p e c u l a t i o n remains to be explored. Acknowledgements. This work was supported i n p a r t by the Veterans A d m i n i s t r a t i o n Research Funds ( P r o j e c t No. 9166) and a grant from the N a t i o n a l I n s t i t u t e s of Health HL 20549. The k i n e t i c s t u d i e s on LPL i n h i b i t i o n by human i n t i m a l g l y c o p r o t e i n were done i n c o l l a b o r a t i o n w i t h P r o f e s s o r Thomas O l i v e c r o n a at the U n i v e r s i t y of Umea, Sweden. I wish to thank Mrs. Betty Blaydes f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Ms. Diane B u t l e r for s e c r e t a r i a l help. A b s t r a c t . This paper briefly reviews the c u r r e n t understanding of a g l y c o p r o t e i n i s o l a t e d from porcine and human aortic i n t i m a . Because the g l y c o p r o t e i n from e i t h e r source inhibits
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
Lipase
211
l i p o p r o t e i n l i p a s e activity, in vitro of post-heparin plasma and of bovine m i l k , it is named " l i p o l i p i n " ( l i p o p r o t e i n l i p a s e in hibitor). The carbohydrate moiety of the porcine molecule (MW 72,000) contains 1 mole each of fucose and s i a l i c a c i d , 2 moles of mannose, 3 moles each of glucose and galactose and 4 moles of N -acetylglucosamine. The s t r u c t u r e of the carbohydrate i s p r e d i c t e d by s e q u e n t i a l a n a l y s i s . Although the molecule from the human a t h e r o s c l e r o t i c i n t i m a is not completely c h a r a c t e r i z e d , it appears that the porcine and human lipolipins share s e v e r a l f e a t u r e s in common: molecular weight, i s o e l e c t r i c p o i n t ( p I 4.3), e l e c t r o p h o r e t i c m o b i l i t y and ratio of carbohydrate c o n s t i t u e n t s . Theref o r e , porcine t i s s u e may serve as a model system t o study the human molecule. K i n e t i c s t u d i e s revealed that human lipolipin decreased both the b a s a l and serum-stimulated a c t i v i t y o f l i p o p r o t e i n l i p a s e . The inhibition was non-competitive w i t h respect to serum. However, hig ed t o relieve the i n h i b i t o r y lipin might be i n v o l v e d in an i n t e r a c t i o n w i t h e m u l s i f i e d lipid denying l i p o p r o t e i n l i p a s e access t o i t s s u b s t r a t e . I f lipolipin is a major c o n s t i t u e n t of t o t a l g l y c o p r o t e i n content in atheros c l e r o t i c a o r t a , the observed higher c o n c e n t r a t i o n of glycoprot e i n s i n a t h e r o s c l e r o t i c plaques as compared t o that i n the uni n v o l v e d regions of the a o r t a may be a reflection of an increased c o n c e n t r a t i o n of l i p o l i p i n i n a t h e r o s c l e r o s i s .
Literature Cited Anderson, J.C. (1976), International Rev. Conn. Tiss. Res., 7, 278. Baig, M.M., and Ayoub, E.M. (1976), Biochemistry 15, 2585. Berenson, G.S., and Fishkin, A.F. (1962), Arch. Biochem. Biophys. 97, 18. Berenson, G.S., Radhakrishnamurthy, B . , Dalferes, E.R., and Srinivasan, S.R. (1971), Human Path. 2, 57. Brockerhoff, H. (1971), J . Biol. Chem. 246, 5828. Buddecke, E. (1960), J . Physiol. Chem. 318, 33. Dicorleto, P.E., and Zilversmit, D.B. (1975), Proc. Soc. Exptl. Biol. Med. 148, 1101. Eisenberg, S., and Levi, R . I . (1975), Adv. Lipid Res. 13, 1. Egelrud, T., and Olivecrona, T. (1972), J . Biol. Chem. 247, 6212. Henson, L.C., and Schotz, M.C. (1975), Biochim. Biophys. Acta 409, 360. Hernell, O., Egelrud, T., and Olivecrona, T. (1975), Biochim. Biophys. Acta 381, 233. I s h i i , M. (1971), Jap. Heart J. 12, 22. Klemer, A . , and Nager, C. (1967), Z. Naturforsch 22, 456. Maier, V . , and Buddecke, E. (1971), Hoppe-Seyler Z. Physiol. Chem. 352, 1338. Manley, G., and Mullinger, R.N. (1967), Brit. J . Path. 58, 529. Muller-Spreer, H.C., Werber, U . , and Voight, K.D. (1960), Klin. Wschr. 38, 28.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
212
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Radhakrishnamurthy, B . , and Berenson, G.S. (1966), J . Biol. Chem. 241, 2106. Radhakrishnamurthy, B . , Fishkin, A . F . , Hubbell, G . J . , and Berenson, G.S. (1964), Arch. Biochem. Biophys. 104, 19. Roberts, B . I . , and Wagh, P.V. (1976), Biochim. Biophys. Acta 439, 26. Roberts, B . I . , White, H . J . , Read, R.C., and Wagh, P.V. (1974), Atherosclerosis 20, 533. Robinson, D.S. (1970), Compr. Biochem. 19, 51. Wagh, P.V. (1975), Adv. Exptl. Med. Biol. 52, 81. Wagh, P.V., and Olivecrona, T. (1978), Atherosclerosis 29, 195. Wagh, P.V., and Roberts, B.I. (1972), Biochemistry 11, 4222. Wagh, P.V., Roberts, B . I . , White, H . J . , and Read, R.C. (1973), Atherosclerosis 18, 83. RECEIVED
February 13, 1978
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12 Carbohydrate Moieties of the Collagens and Collagen-Like Proteins in Health and Disease WILLIAM T. BUTLER Institute of Dental Research, University of Alabama in Birmingham, Birmingham, AL 35294 The fact that interstita proteins contain covalentl for many years. In 1935 Grassmann and Schleich (1) found that hide collagen had equal quantities of firmly bound glucose and galactose, and later studies from this group (2) suggested that the hexoses were bound through O-glycosidic linkages. The stability of the binding of hexoses to collagen was demonstrated by Kühn, et al (3) who found that repeated reprecipitation of citrate-soluble collagen removed all the hexosamine, but only half the hexose. After three reprecipitations, the hexose level was 0.48%, and it remained constant throughout seven additional reprecipitations. This study revealed that hexose, but not hexosamine was an integral part of i n t e r s t i t i a l collagen. Gross, et al (4) examined the amino acid and sugar contents of collagens from a variety of tissues and species. They found that for vertebrate collagens, purification or gelatinization drastically reduced the hexosamine content, but was less effective i n removing hexose. The carbohydrate was identified as glucose and galactose with traces of other hexoses, pentoses and amino sugars. The hexose content was usually less than 1 % for vetebrate and from 3 to 11% i n invertebrate collagens. Similarly Blumenfeld, et al (5) found that ichthyocol (carp swim bladder collagen) contained glucose and galactose but no hexosamine or other sugars. In contrast to the low content of sugars generally found in i n t e r s t i t i a l collagens of vertebrates, the collagen-like proteins of basement membranes contain much higher levels of carbohydrate (6-8). As i n collagen, one type of carbohydrate consists of glucose and galactose units, but heteropolysaccarides with hexoses and hexosamines are also present.
0-8412-0452-7/78/47-080-213$05.00/0 © 1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
214
GLYCOPROTEINS
AND GLYCOLIPIDS IN DISEASE PROCESSES
THE NATURE OF THE LINKAGE P r i o r to 1965 s e v e r a l s t u d i e s on the nature o f the carbohydrate of c o l l a g e n were r e p o r t e d , but the r e s u l t s l e f t many u n c e r t a i n t i e s . The s t u d i e s of B u t l e r and Cunningham (9,10) c l a r i f i e d the nature o f the hexose attached to c o l l a g e n . S t a r t ing w i t h s o l u b l e guinea p i g s k i n c o l l a g e n and d i g e s t i n g to s h o r t e r u n i t s w i t h collagenase and t r y p s i n , glycopeptides were i s o l a t e d . The compositions of the peptides suggested that glucose and galactose might be attached to the 6-hydroxyl group of h y d r o x y l y s i n e . The r e s i s t a n c e of the h y d r o x y l y s i n e to p e r i o d a t e o x i d a t i o n supported t h i s c o n c l u s i o n (9). The nature of the l i n k a g e was c o n c l u s i v e l y shown by the i s o l a t i o n of a glycopeptide w i t h s t o i c h i o m e t r i c amounts of h y d r o x y l y s i n e , glucose, and g a l a c t o s e recovered a f t e r cleavage of the peptide bonds of the c o l l a g e n g l y c o p e p t i d w i t h 2N NaOH t 90° f o 10 hr. The r e s i s t a n c e t o glycosidic; this conclusio supporte y t i o n t h a t m i l d a c i d h y d r o l y s i s (2N HC1, 110°, 30 min.) r e l e a s e d 2 mol of reducing sugar. A l i n k a g e to the e-amino group of h y d r o x y l y s i n e was r u l e d out by the r e a c t i v i t y of t h i s group to f l u o r o d i n i t r o b e n z e n e and by the e l e c t r o p h o r e t i c m o b i l i t y of the glycopeptide. These experiments thus showed that c o l l a g e n cont a i n e d a d i s a c c h a r i d e c o n s i s t i n g of glucose and g a l a c t o s e attached O - g l y c o s i d i c a l l y to h y d r o x y l y s i n e ( G l c - G a l - H y l ) . STRUCTURE OF THE CARBOHYDRATE MOIETY The s t r u c t u r e of the carbohydrate was e l u c i d a t e d by S p i r o (11) u s i n g collagenous components from glomerular basement membranes. A f t e r i s o l a t i o n of a mixture of glycopeptides c o n t a i n ing h y d r o x y l y s i n e - l i n k e d glucose and g a l a c t o s e , he showed t h a t glucose, but not g a l a c t o s e , was r e a d i l y l i b e r a t e d w i t h 0.1 N ^SO^ a t 100° f o r 2-20 h r . Thus glucose was i n an e x t e r n a l p o s i t i o n , w i t h galactose l i n k e d to h y d r o x y l y s i n e . Next a glucose and g a l a c t o s e - c o n t a i n i n g d i s a c c h a r i d e was obtained a f t e r N - a c e t y l a t i o n and m i l d a c i d h y d r o l y s i s of the glycopept i d e s . Glucose was shown to be attached to C-2 of galactose by s t u d i e s employing p e r i o d a t e and g a l a c t o s e oxidase. This l i n k a g e was cleaved by a-glucosidase but not 3-glticosidase. Next G l c Gal-Hyl was i s o l a t e d a f t e r a l k a l i n e h y d r o l y s i s of g l y c o p e p t i d e s ; the anomeric c o n f i g u r a t i o n of the g a l a c t o s e l i n k a g e to hydroxyl y s i n e was shown to be 3 by i t s s u s c e p t i b i l i t y to 3 g a l a c t o s i d a s e . These experiments thus i n d i c a t e d that G l c - G a l - H y l had the s t r u c ture 2-0-a-D-glucopyranosy1-0-3-D-galac t o p y r a n o s y l h y d r o x y l y s i n e (Figure 1). A s i m i l a r s t r u c t u r e was proposed by K e f a l i d e s (12). The s t r u c t u r e of G l c - G a l - H y l i n c o l l a g e n s from v e r t e b r a t e and i n v e r t e b r a t e sources has been shown to be the same as that found i n basement membranes. Thus Cunningham and Ford (13) observed that glucose was s p l i t from the d i s a c c h a r i d e of guinea -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
Moieties
of the
Collagens
215
p i g s k i n c o l l a g e n by m i l d a c i d h y d r o l y s i s , w h i l e galactose r e mained attached. They a l s o reported f i n d i n g g a l a c t o s y l h y d r o x y l y s i n e (Gal-Hyl) a f t e r a l k a l i n e h y d r o l y s i s of s k i n c o l l a g e n . Using the amino a c i d a n a l y z e r , S p i r o (14) introduced a separat i o n technique f o r q u a n t i t a t i o n of G l c - G a l - H y l and Gal-Hyl. Chromatography o f a l k a l i n e h y d r o l y s a t e s of s e v e r a l c o l l a g e n samples showed that they contained the same mono- and d i s a c c h a r i d e u n i t s l i n k e d to h y d r o x y l y s i n e as basement membranes. Katzman, jet a l (15) u t i l i z e d G l c - G a l - H y l i s o l a t e d from the sponge and showed that the s t r u c t u r e was that proposed f o r basement membranes by S p i r o (11). The h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e i s o l a t e d from sea anenome, sea cucumber, and bovine cornea was shown to have an i d e n t i c a l s t r u c t u r e by a v a r i e t y of techniques (15). Since these e a r l y s t u d i e s , G l c - G a l H y l and Gal-Hyl have been shown to occur i n a v a r i e t y of c o l l a gen and c o l l a g e n - l i k e p r o t e i n s OCCURRENCE IN THE DIFFEREN The d i s c o v e r y that c a r t i l a g e contained a type o f c o l l a g e n d i s t i n c t from t h a t o f s k i n and bone (16) ushered i n a new e r a i n c o l l a g e n biochemistry. We now understand that a t l e a s t three c o l l a g e n s w i t h unique p r o p e r t i e s e x i s t i n the e x t r a c e l l u l a r m a t r i x of connective t i s s u e s (Table I ) . For more d e t a i l s conc e r n i n g these c o l l a g e n s , the reader i s r e f e r r e d t o the review by M i l l e r (17). S c i e n t i s t s have long recognized t h a t the basement membranes c o n t a i n collagenous components (6,18,19). A f i n a l r e s o l u t i o n of the s t r u c t u r e s o f the molecular species present i n basement membranes has not been accomplished. A t l e a s t one o f these appears to have a t r i p l e - h e l i c a l conformation r e f e r r e d t o as [ a l ( I V ) ] 3 (20). However i t i s apparent that s e v e r a l other s t r u c t u r e s are present (21-23). A l l three o f the i n t e r s t i t i a l c o l l a g e n s c o n t a i n hexose attached to h y d r o x y l y s i n e , but type I I c o l l a g e n has a s i g n i f i c a n t l y greater q u a n t i t y of h y d r o x y l y s i n e and o f h y d r o x y l y s i n e l i n k e d carbohydrate (Table I ) . I n general the collagenous p o r t i o n of basement membranes c o n t a i n much higher l e v e l s o f h y d r o x y l y s i n e and of the a s s o c i a t e d glucose and g a l a c t o s e moieties. I t should be noted that other h y d r o x y p r o l i n e - c o n t a i n i n g p r o t e i n s w i t h c o l l a g e n - l i k e ( i . e . Gly-X-Y repeating) sequences e x i s t . For example, the complement p r o t e i n C l q contains s e v e r a l g l y c o s y l a t e d hydroxylysines i n a c o l l a g e n - l i k e sequence (24). The exact l o c a t i o n s o f s e v e r a l o f the carbohydrate m o i e t i e s of the collagens has been documented during the s t u d i e s on the primary s t r u c t u r e s o f the v a r i o u s a chains. One s i t e of g l y c o s y l a t i o n common to the f o u r chains of the three i n t e r s t i t i a l c o l l a g e n s i s Hyl-87 (Figure 2). The amino a c i d sequences around t h i s s i t e are s i m i l a r i n these chains (25-29). The occurrence of the d i s a c c h a r i d e a t t h i s s i t e suggests that i t performs some
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2
[al(II)]
[al(III)]
Several
II
III
IV Basement Membranes (Glomerulus, e t c . )
Ubiquitous ( E s p e c i a l l y Prominent i n F e t a l S k i n , Arteries, Cirrhotic Liver)
Cartilage, Intervetebral D i s c , Notocord
Ubiquitous ( S k i n , Bone, Tendon Dentin, etc.)
Tissue D i s t r i b u t i o n
40-50
5-8
20-25
5-15
b
55
2
b
15-30
2.5
a
Hexose Content (Residues/1000)
Data f o r the c o l l a g e n - l i k e p o r t i o n of glomerular basement membranes. Taken from r e f e r e n c e 19.
Glucose and galactose
3
[al(I)] o2
I
3
Molecular Composition
Type
Hydroxylysine Content (Residues/1000)
TABLE I : S t r u c t u r e s and C h a r a c t e r i s t i c s of I n t e r s t i t a l Collagens and C o l l a g e n - L i k e P r o t e i n s of Basement Membranes
BUTLER
Carbohydrate
Moieties of the
Collagens
CH OH 2
HC* V^~° OH
"^v
6H -NH © 2
3
®H N-CH-C-O© 3
CH OH
^
2
HO/KJ VOH
° \ Hy H
" /
CH OH 2
»yfr—°\H/°
g
HO
old)
2
Q
CH2-NH3®
Figure I. The structures of the glucose- and galactose-containing units isolated from the collagens d collagen-like proteins. (A) / ? - D galactopyranosylhydroxylsine (Gal an
hyl),
H/^ OH
(B)
2-O-a-D-glucopyranosyl
O - /? - D- galactopyranosylhydroxyly sine (Glc-Gal-Hyl),
- GLY -
Glc - Gal
a2
- GLY - L E U - HYP - GLY - P H E - HYL - GLY - I L E - ARG 1
Glc - Gal
a I (II)
- GLY - L E U - HYP - GLY - V A L - HYL - GLY - H I S - ARG -
Glc - Gal
a l(III)
- GLY - PHE - HYP - GLY - MET - H Y L - G L Y - H I S - ARG 1
Glc - Gal
Figure 2. Amino acid sequences around the disaccharide-bearing hydroxylysines common to types J, II, and III collagens
old)
174 - G L Y - A L A . A L A - G L Y - A L A - L Y S - GLY - G L U - A L A -
al(III)
- GLY - SER - HYP - GLY - A L A - L Y S - GLY - GLU - V A L -
ol(II)
- G L Y - A L A - HYP - G L Y - A L A - H Y L - G L Y - GLU - A L A •
Gal
o2
- G L Y - A L A - HYP - GLY - PRO - HYL - G L Y - GLU - L E U 1
Gal
Figure 3.
The amino acid sequences of a chains around the monosaccharide-bearing hydroxylysine of a2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
218
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
f u n c t i o n a l r o l e v i t a l to the three collagens (e.g. i n c r o s s l i n k i n g ) . The a2 chain of type I c o l l a g e n has a Gal-Hyl at p o s i t i o n 174 (26,27) not found i n a l ( I ) or a l ( I I I ) but present i n a l ( I I ) (Figure 3). Although these appear to be the major s i t e s of carbohydrate i n a l ( I ) , a2 and a l ( I I I ) , other p o s i t i o n s may be p a r t i a l l y g l y c o s y l a t e d . For i n s t a n c e s u b i n t e g r a l l e v e l s of Gal-Hyl are found near the COOH-terminus of a l ( I ) i n c a l f s k i n c o l l a g e n (26). As s t a t e d b e f o r e , the a l ( I I ) chain of c a r t i l a g e c o l l a g e n has many more s i t e s of carbohydrate attachment than the above. Studies on the covalent s t r u c t u r e of bovine and c h i c k a l ( I I ) chains (28,30,31) from t h i s l a b o r a t o r y have a l r e a d y l o c a t e d 13 g l y c o s y l a t e d hydroxylysines (Figure 4 ) , although only about 60% of the sequence i s known. These h y d r o x y l y s i n e s are not confined to any one area but occur throughout the chain. Comparison of the p o s i t i o n s of these g l y c o s y l a t e d h y d r o x y l y s i n e s w i t h homolo gous sequences i n a l ( I occupied by l y s i n e i n th chains have s i m i l a r amino a c i d sequences w i t h p o t e n t i a l s i t e s f o r carbohydrate attachment, but only f o r a l ( I I ) are the postt r a n s l a t i o n a l m o d i f i c a t i o n s ( i . e . h y d r o x y l a t i o n and g l y c o s y l a t i o n ) made. I w i l l r e t u r n to p o s s i b l e explanations f o r the h i g h l e v e l of Glc-Gal-Hyl and Gal-Hyl i n a l ( I I ) i n a l a t e r s e c t i o n . BIOSYNTHETIC ATTACHMENT OF HEXOSE TO PROCOLLAGEN a CHAINS Collagen i s synthesized w i t h i n connective t i s s u e c e l l s by the u s u a l p r o t e i n s y n t h e t i c mechanism 032) but has s e v e r a l f e a t u r e s which are unique (Figure 5). The i n i t i a l form of the three a chains are elongated, compared to that found i n f i b r i l l a r c o l l a g e n (33-36). These " e x t e n s i o n s " which occur on the NH2~and COOH-terminal ends of the c h a i n s , are cleaved from the molecules during or a f t e r e x i t from the c e l l . During and a f t e r the t r a n s l a t i o n of these p r o c o l l a g e n chains on ribosomal complexes, seve r a l p o s t - t r a n s l a t i o n m o d i f i c a t i o n s take p l a c e . L y s y l and p r o l y l residues are hydroxylated to form h y d r o x y l y s y l and h y d r o x y p r o l y l r e s i d u e s , and galactose and glucose m o i e t i e s are attached to c e r t a i n of the h y d r o x y l y s i n e s . Next the three chains of a p r o c o l l a g e n u n i t a s s o c i a t e , i n t e r c h a i n d i s u l f i d e bonds i n the COOH-terminal extensions are formed, and f o l d i n g i n t o a t r i p l e - c h a i n c o l l a g e n h e l i x takes p l a c e . I t i s obvious that the attachment of galactose and glucose to c o l l a g e n must be preceded by h y d r o x y l a t i o n of l y s y l r e s i d u e s . This r e a c t i o n i s c a t a l y z e d by l y s y l hydroxylase (32,29,40), an enxyme which i s s i m i l a r i n many respects to p r o l y l hydroxylase. Each of these mixed f u n c t i o n oxidases r e q u i r e s molecular oxygen, a - k e t o g l u t a r a t e , ascorbate and f e r r o u s i r o n (Figure 6) and each c a t a l y z e s h y d r o x y l a t i o n of r e s i d u e s i n the Y p o s i t i o n of the r e p e a t i n g Gly-X-Y c o l l a g e n sequence (37), though l y s y l hydroxyl a s e w i l l apparently a l s o a c t upon l y s i n e s i n the X p o s i t i o n (40).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
Moieties
of the
219
Collagens
al(l) Bovine al(ll) Bovine a 2 Bovine
87 GLY - LEU - HYP. - GLY - MET - HYL - GLY - HIS - ARG - GLY - PHE - SER GLY - LEU - HYP • GLY - VAL - HYL - GLY - HIS - ARG - GLY - THR - HYP GLY - LEU - HYP - GLY - PHE - HYL - GLY - ILE - ARG - GLY - HIS - ASN
al(l) Bovine al(ll) Bovine a 2 Bovine
99 GLY - LEU - ASP - GLY - ALA - LYS - GLY - ASP - ALA - GLY - PRO - A L A GLY - LEU - ASP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - ALA - HYP GLY - LEU - ASP - GLY - LEU - THR - GLY - GLN - HYP - GLY - ALA - HYP
al(l) Bovine al(ll) Bovine a 2 Bovine
108 GLY - PRO - ALA - GLY - PRO - LYS - GLY - GLU - HYP - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - SER - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - HYP - GLY - ALA - HYP
al(l) Rat a 1(11) Bovine a 2 Bovine
174 GLY - ALA - ALA - GLY - ALA - LYS - GLY - GLU '- ALA - GLY - PRO - GLN GLY - ALA - HYP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - PRO GLY - ALA - HYP - GLY - PRO - HYL - GLY - GLU - LEU - GLY - PRO - VAL
al(l) Rot a 1(11) Bovine a 2 Bovine
GLY - GLN - HYP - GLY GLY - ILE - HYP - GLY GLY - LEU - HY
al(l)Rat al(l 1) Bovine a 2 Bovine
GLY - ALA GLY - PRO GLY - ALA
al(l) Bovine a 1(11) Bovine a 2 Chick
GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY
a 1(1) Bovine al(ll)Bovine a 2 Chick
GLY - LYS GLY - LY