Sema4D

CD100/Sema4D Atsushi Kumanogoh and Hitoshi Kikutani* Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan * corresponding author tel: +81-6-6879-8363, fax: +81-6-6875-4465, e-mail: [email protected] DOI: 10.1006/rwcy.2002.0329.

SUMMARY

Alternative names

CD100 is a 150 kDa transmembrane protein that belongs to the semaphorin family. Many members of the semaphorin family have been identified as axonal guidance factors during neuronal development. CD100 is the first semaphorin member shown to have physiological roles and expression in the immune system. CD100 utilizes two types of receptors in different tissues. Plexin-B1 has been shown to be the receptor of CD100 in nonlymphoid tissues, while CD72 has been identified to be a lymphocyte receptor of CD100 in lymphoid tissues. CD100 functions as a ligand in the immune system through CD72 to augment the proliferation and antibody production of activated B cells and to promote the activation and maturation of antigen-presenting dendritic cells.

Sema4D/SEMA4D, M-Sema G (mouse), and C-Coll-4 (chick).

BACKGROUND

Discovery CD100 was originally identified as a 150 kDa surface antigen recognized by monoclonal antibodies (mAb) raised against human T cells (Herold et al., 1995). In 1996, molecular cloning revealed that CD100 is a transmembrane-type semaphorin member, which provided the first evidence that semaphorins are expressed in the immune system (Hall et al., 1996). Mouse CD100 was independently cloned from the brain and has been shown to be expressed in lymphoid tissues as well (Furuyama et al., 1996). Mouse CD100 was also cloned as one of molecules induced in CD40-stimulated B cells (Kumanogoh et al., 2000).

Cytokine Reference

Structure The semaphorins are defined by the presence of a common signature domain, `sema domain', at their N-terminal end, which is a stretch of approximately 500 amino acids (Yu and Kolodkin, 1999). Based on their additional structural features, semaphorins have been divided into eight classes. CD100 belongs to the class IV of the semaphorin family and consists of an N-terminal signal sequence followed by a sema domain, an Ig-like domain, a lysine-rich stretch, a hydrophobic transmembrane region and a cytoplasmic tail (Kumanogoh and Kikutani, 2001). The extracellular region of CD100 contains several potential N-linked glycosylation sites and conserved cysteines in the sema domain. In the cytoplasmic region of CD100, there is a consensus site for tyrosine phosphorylation and multiple consensus sites for serine phosphorylation. In both human and mouse, a soluble isoform of 120 kDa is released from activated lymphocytes as a proteolytic product of a transmembrane CD100 (Elhabazi et al., 1997; Wang et al., 2001).

Main activities and pathophysiological roles Although the biological activities of CD100 have been ascribed to both a receptor and a ligand, it has been

Copyright # 2002 Published by Elsevier Science Ltd

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Atsushi Kumanogoh and Hitoshi Kikutani

clearly shown that mouse CD100 plays a physiological role as a ligand in immune regulation. Mouse recombinant soluble CD100 or transfectants expressing transmembrane-type CD100 can augment proliferation and antibody production of CD40stimulated mouse B cells (Kumanogoh et al., 2000). Human CD100-expressing transfectants also enhance human B cell activation (Hall et al., 1996). CD100 enhances the activation and maturation of dendritic cells, which is necessary to enable them to interact with antigen-specific T cells. These functions of CD100 have been confirmed by in vivo and in vitro experiments with CD100 knockout and transgenic mice. In addition, elevated serum levels of soluble CD100 are correlated with autoantibody titers of MRL/lpr mice (Wang et al., 2001). Furthermore, CD100-deficient mice are resistant to experimental autoimmune encephalomyelitis (Kumanogoh et al., 2002). These observations suggest that CD100 may contribute to pathological immune responses, particularly autoimmune responses.

GENES AND GENE REGULATION

Accession numbers Human: XM-108898 Mouse: NM-13660

Chromosome location Human 9q22-q31

Cells and tissues that express the gene CD100 mRNA has been detected in large quantities in a broad range of tissues, including embryonic and adult brain, kidney, and heart (Furuyama et al., 1996; Hall et al., 1996). In mice, CD100 mRNA is detected throughout the embryonic nervous system, with considerable expression in the cortical plate and dorsal root ganglia. In addition, the embryonic thymus exhibits very marked expression and the embryonic lung and kidney show moderate expression. CD100 mRNA is detectable in the brain, kidney, thymus, spleen, lung, heart, and bone marrow in both adult human and mouse.

PROTEIN

Accession numbers Human: XP-108898 Mouse: NP-038688

Sequence See Figures 1 and 2.

Description of protein Only the primary sequence of CD100 is known. CD100 is a member of the semaphorin family. There is 81% amino acid identity between human and mouse CD100 (Furuyama et al., 1996; Hall et al., 1996). CD100 has a large conserved sema domain in its extracellular region as well as the other family members. In addition, there is an Ig-like domain between sema and transmembrane domains. A mutational analysis of human CD100 has demonstrated that the cysteine residue at position 679 in the

Figure 1 Amino acid sequence for human CD100. MRMCTPIRGL TLYIGAREAV TSLYVCGTNA FLGSEPIISR EYEFVFRVLI SPGLKVPVFY PVPKPRPGAC TQIVVDRTQA KKGNRFVYAG GLIQEMSGDA YGLMGRKNLL VQTEGSRIAT HSEKTMYLKS DFCDREQSLK ARDKPFDVKC

LMALAVMFGT FAVNALNISE FQPACDHLNL NSSHSPLRTE PRIARVCKGD ALFTPQLNNV IDSEARAANY LDGTVYDVMF SNSGVVQAPL SVCPDKSKGS IFNLSEGDSG KVLVASTQGS SDNRLLMSLF ETLVEPGSFS ELKFADSDAD

Figure 2 MRMCAPVRGL TLYVGAREAV TSLYVCGTNA FLGSEPIISR EYEFVFKLMI APGLKEPVFY PVPTPRPGAC TQIVVDRTQA KKGRKFVYAG GWIQDMSGDT YGFVGRKHLL VQTEGSKITS SEKTVYLKSS FSDLEQSVKE RDKPFDVKCE

AMAFAPIPRI KQHEVYWKVS TSFKFLGKNE YAIPWLNEPS QGGLRTLQKK GLSAVCAYNL TSSLNLPDKT VSTDRGALHK AFCGKHGTCE YRQHFFKHGG VYQCLSEERV SPPTPAVQAT LFFFVLFLCL QQNGEHPKPA GD

TWEHREVHLV EDKKAKCAEK DGKGRCPFDP FVFADVIRKS WTSFLKARLI STAEEVFSHG LQFVKDHPLM AISLEHAVHI DCVLARDPYC TAELKCSQKS KNKTVFQVVA SSGAITLPPK FFYNCYKGYL LDTGYETEQD

QFHEPDIYNY GKSKQTECLN AHSYTSVMVD PDSPDGEDDR CSRPDSGLVF KYMQSTTVEQ DDSVTPIDNR IEETQLFQDF AWSPPTATCV NLARVFWKFQ KHVLEVKVVP PAPTGTSCEP PRQCLKFRSA TITSKVPTDR

SALLLSEDKD YIRVLQPLSA GELYSGTSYN VYFFFTEVSV NVLRDVFVLR SHTKWVRYNG PRLIKKDVNY EPVQTLLLSS ALHQTESPSR NGVLKAESPK KPVVAPTLSV KIVINTVPQL LLIGKKKPKS EDSQRIDDLS

Amino acid sequence for mouse CD100.

FLALVVVLRT FAVNALNISE FQPTCDHLNL NSSHSPLRTE PRVARVCKGD AVFTPQLNNV IDSEARAANY LDGTFYDVMF SNSGVVQAPL SSCLDKSKES IFNLSDGDSG KMPVGSTQGS DNRLLMSLLL TLVEPGSFSQ LKFADSDADG

AVAFAPVPRL KQHEVYWKVS TSFKFLGKSE YAIPWLNEPS QGGLRTLQKK GLSAVCAYTL TSSLNLPDKT ISTDRGALHK AFCEKHGSCE FNQHFFKHGG VYQCLSEERV SPPTPALWAT FIFVLFLCLF QNGDHPKPAL D

TWEHGEVGLV EDKKSKCAEK DGKGRCPFDP FVFADVIQKS WTSFLKARLI ATVEAVFSRG LQFVKDHPLM AVILTKEVHV DCVLARDPYC TAELKCFQKS RNKTVSQLLA SPRAATLPPK SYNCYKGYLP DTGYETEQDT

QFHKPGIFNY GKSKQTECLN AHSYTSVMVG PDGPEGEDDK CSKPDSGLVF KYMQSATVEQ DDSVTPIDNR IEETQLFRDF AWSPAIKACV NLARVVWKFQ KHVLEVKMVP SSSGTSCEPK GQCLKFRSAL ITSKVPTDRE

SALLMSEDKD YIRVLQPLSS GELYSGTSYN VYFFFTEVSV NILQDVFVLR SHTKWVRYNG PKLIKKDVNY EPVLTLLLSS TLHQEEASSR NGELKAASPK RTPPSPTSED MVINTVPQLH LLGKKTPKSD DSQRIDELSA

CD100/Sema4D 3 sema domain is necessary for homodimerization of CD100 (Delaire et al., 2001; Elhabazi et al., 2001), which has been shown to be essential for the biological function of CD100.

Posttranslational modifications The extracellular region of CD100 has several potential N-linked glycosylation sites. Indeed, Nglycosidase treatment of recombinant mouse CD100 proteins reduces its molecular weight (Kumanogoh et al., 2000). Although CD100 is a semaphorin member of the transmembrane type, both human and mouse CD100 have been shown to be proteolytically cleaved into a 120 kDa soluble form. This cleavage has been suggested to be metalloproteinase-dependent (Elhabazi et al., 2001). Soluble mouse CD100, naturally released from activated lymphocytes, has a biological activity similar to that of transmembrane CD100 (Wang et al., 2001).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce The expression of CD100 is detected on most hematopoietic cells, except immature bone marrow cells, RBC, and platelets (Kumanogoh and Kikutani, 2001). In particular, the expression of CD100 is abundantly expressed on the surface of T cells. Its expression is relatively weak on B cells, macrophages, and dendritic cells. However, once activated, its expression is significantly upregulated on these cells.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Various stimuli have been reported to induce upregulation of CD100. In T cells, the expression of CD100 is upregulated after the treatment with antiCD3, ConA, or PHA (Herold et al., 1995; Delaire et al., 1998). In B cells and antigen-presenting cells, CD100 expression is upregulated after stimulation with anti-CD40 and LPS (Kumanogoh et al., 2000; Kumanogoh and Kikutani, 2001).

RECEPTOR UTILIZATION Two types of receptors with distinct binding affinities, plexin-B1 and CD72, have been identified as CD100/ Sema4D receptors. Plexin-B1, which is widely expressed at prominent levels in the fetal brain and kidney, is a receptor that has been shown to have a high affinity (Kd  1  10 9 M) for CD100 (Tamagnone et al., 1999). Human CD100 stimulation has been shown to enhance the interaction between the cytoplasmic region of plexin-B1 and a small GTPase, Rac (Driessens et al., 2001). However, little is known concerning the physiological importance of CD100±plexin-B1 interactions. On the other hand, CD72 has been demonstrated to be a receptor on lymphocytes for CD100 with low affinity (Kd  3  10 7 M) through expression cloning (Kumanogoh et al., 2000). CD72 is a 45 kDa type II transmembrane protein that belongs to the C-type lectin family (Nakayama et al., 1989; Von Hoegen et al., 1990). CD72 is not only expressed on the surface of B cells but also expressed on antigen-presenting cells, including dendritic cells, macrophages, and some subpopulations of T cells. Although crosslinking of CD72 by its agonistic mAbs or CD100 enhances B cell activation, CD72 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain and associates with a tyrosine phosphatase, SHP-1, which is a common feature of inhibitory receptors (Adachi et al., 1998). Interestingly, CD100 induces tyrosine dephosphorylation of CD72 and SHP-1 dissociation. This may be one of major mechanisms by which CD100 enhances B cell activation through CD72 (Kumanogoh et al., 2000). It has been reported that CD100 enhances the interaction between the cytoplasmic region of plexinB1 and a small GTPase, Rac (Driessens et al., 2001). However, the physiological role of CD100±Plexin-B1 interactions remains unknown. CD100 has a cytoplasmic region, which has been reported to be capable of signal transduction. In the cytoplasmic region of CD100, there is a consensus site for tyrosine phosphorylation and multiple consensus sites for serine phosphorylation. Associated serine kinase activities have been reported to regulate the generation of soluble CD100 (Elhabazi et al., 1997). Tyrosine phosphatases, including CD45, have been also reported to associate with CD100 (Herold et al., 1996). CD100/Sema4D-deficient mice have functional defects in lymphoid tissues where CD72 is expressed, but not in other tissues where plexin-B1 is abundantly expressed (Shi et al., 2000). Therefore, the

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interaction of CD100/Sema4D with CD72 appears to play a nonredundant role in the immune system.

IN VITRO ACTIVITIES

In vitro findings Functions as a Receptor Using mAbs specific for human CD100, CD100 has been shown to signal through its cytoplasmic domain. Antibody-mediated crosslinking of human CD100 has been shown to enhance the proliferation of T cells in the presence of submitogenic doses of anti-CD3 or anti-CD2 mAbs (Herold et al., 1995, 1996). The cytoplasmic region of CD100 has been shown to be associated with serine/threonine kinase activities (Elhabazi et al., 1997). CD45, a cell-surface protein tyrosine phosphatase (PTP), has been reported to be associated with CD100 in human T cells (Herold et al., 1996). It has been reported that a switch in the types of PTP associated with human CD100 occurs at the terminal B cell differentiation stages (Billard et al., 2000). However, it is still not clear whether CD100 is involved in physiological or pathological immune responses as a signal-transducing receptor. Functions as a Ligand Human CD100-expressing transfectants have been shown to promote the aggregation and survival of B cells (Hall et al., 1996). Also, mouse CD100expressing transfectants and recombinant mouse CD100 proteins have been shown to enhance CD40induced B cell responses including proliferation and immunoglobulin production (Kumanogoh et al., 2000). In addition to the important roles of CD100 in B cell responses, it has been demonstrated that CD100 is also involved in the maturation of dendritic cells (DCs) in the context of costimulatory molecule expression and IL-12 production as a consequence of maturation.

IN VIVO ACTIVITIES

antigens are severely impaired (Shi et al., 2000). In contrast, in CD100-overexpressing transgenic mice, these responses are enhanced (Watanabe et al., 2001). Collectively, these findings indicate a crucial role of CD100 in humoral immune responses. Cellular Immunity In addition to humoral immunity, CD100 plays an important role in cellular immunity. The generation of antigen-specific T cells is severely impaired in CD100-deficient mice but is enhanced in CD100transgenic mice (Shi et al., 2000; Watanabe et al., 2001). The effect of CD100 on the ability of DCs to stimulate antigen-specific T cells in vivo was also tested. Bone marrow-derived DCs were pulsed with OVA protein, treated with CD100 plus anti-CD40, and then injected into mice. Consequently, the generation of antigen-specific T cells and DTH responses were significantly enhanced in naõÈ ve mice injected with such treated DCs, although DTH response in the mice injected with DCs that were pulsed with OVA and stimulated with anti-CD40 alone was not observed. Collectively, these results indicate that CD100 is involved in cellular immunity by enhancing the activities of DCs.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects In sera of normal mice, soluble forms of CD100 cannot be detected. In sera of mice immunized with TD antigens, soluble forms of CD100 become detectable and the levels of sCD100 correlated well with the titers of antigen-specific antibodies (Wang et al., 2001). In MRL/lpr mice, the levels of sCD100 correlate well with the titers of autoantibodies.

Humoral Immunity

Role in experiments of nature and disease states

Administration of soluble mouse CD100 protein has been demonstrated to accelerate in vivo antibody production against T cell-dependent (TD) antigens (Kumanogoh et al., 2000). In CD100-deficient mice, antigen-specific antibody production against TD

Experimental autoimmune encephalomyelitis is induced by immunization with MOG peptide. CD100-deficient mice are resistant to the development of EAE, suggesting the involvement of CD100 in pathological immune responses.

CD100/Sema4D 5

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