CD6 recognizes the neural adhesion molecule ... - Wiley Online Library

Protein Science (1997), 61768-1770. Cambridge University Press. Printed in the USA. Copyright 0 1997 The Protein Society

FOR THE RECORD

CD6 recognizes the neural adhesion molecule BEN

JOHN E. SKONIER,' MICHAEL A. BOWEN,' ALETANDRO ARUFF0,2 AND JURGEN BAJORATH'

'

Bristol-Myers Squibb Pharmaceutical Research Institute (BMS-PRI), 3005 First Avenue, Seattle, Washington 98 I21 'BMS-PRI and Department of Biological Structure, University of Washington, Seattle, Washington 98195

Abstract: CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6-ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with -10-fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAMand BEN fusion proteins containing the full-length extracellular regions are tested. Homotypic interactions between full-length forms are likely to account for these observations. The identified cross-species interaction between CD6 and BEN suggests that CD6-ligand interactions are highly conserved. Keywords: binding avidity; CD166; CD6; cell surface receptorligand interaction; residue conservation; specificity

CD6 is a cell surface receptor of the scavenger receptor cysteinerich (SRCR) protein family (Aruffo et al., 1991; Resnick et al., 1994), while the human CD6 ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) (Bowen et al., 1995) belongs to the immunoglobulin (Ig) superfamily (Williams & Barclay, 1988). CD6 is expressed on thymocytes, some B cells, and mature T cells (Gangemi et al., 1989; Aruffo et al., 1991). CD6 mRNA has also been detected in the brain. ALCAM is expressed by thymic epithelial cells, activated B and T cells, monocytes (Bowen et al., 1995; Patel et al., 1995), and in the brain (Mayer et al., 1990). CD6 and CD6-ALCAM interactions have been implicated in the regulation of T cell function (Morimoto et al., 1988; Gangemi et al., 1989)andthymocyte-thymicepithelial cell adhesion(Bowen et al., 1995). Whether CD6-ligand interactions play a role in the nervous system, as suggested by the detection of both CD6 and ALCAM in the brain, is currently unknown. Both CD6 and ALCAM are type I transmembrane proteins. The extracellular region of CD6 consists of three SRCR domains (Aruffo et al., 1991). The extracellular region of ALCAM is composed of five Ig domains (Bowen et al., 1995). The twoN-terminal domains show V(ariab1e)-set sequence characteristics, while the other doReprint requests to: Jurgen Bajorath, BMS-PRI, 3005 First Avenue, Seattle, Washington 98121; e-mail: [email protected].

mains are C(onstant)-like (Williams & Barclay, 1988). The domain organization of CD6 and ALCAM is schematically shown in Figure 1. Studies with soluble recombinant forms of CD6 and ALCAM have shown that the membrane-proximal SRCR domain of CD6 specifically binds the N-terminal Ig domain of ALCAM in a 1 :1 interaction (Fig. 1) and that the presence of other extracellular domains is not required for binding (Whitney et al., 1995; Bowen et al., 1996) ALCAM and the chicken neural adhesion molecule BEN (DMGRASP) (Bums et al., 1991; Pourquie et al., 1992), which supports neurite growth, share equivalent extracellular domain organization and significant sequence homology (Fig. 2a). The N-terminal Ig domains of ALCAM and BEN display -85% sequence identity, while the second domains are -72% identical. Here we have investigated the possibility of a cross-species interaction between CD6 and BEN and have compared binding characteristics with ALCAM. Sequence comparison shows that the N-terminal CD6 binding domains of human and mouse (Bowen etal., 1997) ALCAM differ at only six positions, whereas human ALCAM and BEN differ at 16 positions (Fig. 2A). The residue differences were mapped on a

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Fig. 1. Schematic representation of the extracellular domain organization and interaction of CD6 and ALCAM. In the text, ALCAM domains are termed Dl (Ig) to D5 beginning from the N-terminus. The membraneproximal extracellular domain of CD6 is labeled SRCR.

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ITWYRNGKVLQPVEGEVAILFKKEIDPGTQLYTVTSSLEYKTTRSNIQMPFTCSVTYYGPSGQKTIYSEQ ..........h.l..a.v.i. ...m..v .....m ..t. kad.. ...................... .... k..r......ev...n.r.venrs.g.f.m....q.mp.kedanak...i...h........q..p

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Fig. 2. Analysis of sequence homology. A: Sequence comparison of the two N-terminal extracellular domains of human (h) and mouse (m) ALCAM and chicken(c) BEN. 4106 marks theend of the first domain (Dl). Dots indicate identical residues and lowercase letters show residue differences. Exclamation marks residues in human ALCAM which, when mutated, affect CD6 binding. B: Mapping of non-conserved residues on a molecular model (solid ribbon) of the N-terminal Ig domain of human ALCAM. The alpha carbon positions of residues that are important for CD6 binding are. shown as red spheres. On the left, positions that differ in human and mouse ALCAM are shown in yellow. On the right, residue differences in human ALCAM and BEN are shown in gold. Selected residues are numbered.

molecular model ofthe N-terminal domain ofhumanALCAM (Bajorath et al., 1995) relative to residues whose mutations affect CD6 binding but not structural integrity (Skonier et al., 1996). Residues not conserved in mouse and human ALCAM map to the @sheet surface opposite the CD6 binding site, which is strictly conserved in these molecules (Fig. 2B), consistent with the finding that human CD6 binds both human and mouse ALCAM (Bowen et al., 1997). In contrast, residue differences in human ALCAM and BEN are scattered throughout the domain. Some nonconservative residue replacements closely map to residues important for CD6 binding, indicating that the putative CD6 binding surface, which is strictly conserved in ALCAM,is not conserved in BEN.

To test for CD6-BEN interactions and to compare CD6-BEN/ ALCAM binding, Ig fusion proteins including different extracellular domain combinations (Fig. 1) were prepared. ALCAM Dl-Ig includes only the N-terminal domain fusedto Ig constant regions, while ALCAM DlD2-Ig and DlD2D3-Ig include the first two and three extracellular domains, respectively. In a BEN Dl/ALCAM D2 hybrid Ig fusion protein, the N-terminal domain of BEN was combined with the second extracellular domain ofALCAM. In addition, Ig fusion proteins including all five extracellular domains of BEN or ALCAM (full-length forms) were prepared. These fusion proteins were tested for binding to full length CD6-Ig. The results of the CD6 binding experiments are summarized in Fig-

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ure 3. Experimental details regarding the generation of fusion proteins and the binding assays are provided in the legend of Figure 3. In accordance with previous findings, the binding of ALCAM D l - and DlDZIg to CD6 was similar in our assay (not shown). Figure 3A demonstrates that BEN DlD2-Ig specifically bound to CD6, albeit with at least 10-fold lower avidity. This discrepancy is probably a consequence of residue differences in the CD6 binding region (Fig. 2B).The BEN Dl/ALCAM D2 hybrid bound CD6

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like BEN D1D2 (Fig. 3A). This observation confirms that ALCAM D2 does not significantly contribute to CD6 binding. We also compared the binding of full-length ALCAM and full-length BEN to CD6. In contrast to the different binding characteristics of the ALCAM and BEN D1D2 fragments, the full length forms bound CD6 at very similar levels (Fig. 3B). These apparent differences in binding avidity may be explained when interactions between domains not critical for receptor binding are considered. BEN is known to form homotypic interactions (Pourquie et al., 1992). Similarly, the addition of other ALCAM domains to ALCAM Dl-Ig leads to increasing oligomerization states in solution (Skonier et al., 1996). ALCAM and BEN domains not critical for CD6 binding are thus likely to contribute to oligomerization on the cell surface that modulates the avidity of the receptor-ligand interaction. In conclusion, we have demonstrated that CD6 recognizes the neural adhesion molecule BEN. The identification of the crossspecies interaction between CD6 and BEN suggests that CD6ligand interactions are highly conserved and may play a role in both the immune and nervous systems.

References

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Aruffo A, Melnick MB. Linsley PS, Seed B. 1991. The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristicof a new family of cell surface and secreted proteins. J Exp Med 174949-952. 0.1 1 10 100 1000 10000 Bajorath J, Bowen MA, Aruffo A. 1995. Molecular model of the N-terminal receptor binding domain of the human CD6 ligand ALCAM. Prorein Sci PROTEIN (nglml) 4:1644-1647. Bowen MA, Bajorath J, Siadak AW, Modrell B, Malacko AR, Marquardt H, Nadler SG, Aruffo A. 1996. The amino-terminal immunoglobulin-like do1.6 main of activated leukocyte cell adhesion molecule binds specifically to the membrane-proximal scavenger-receptor cysteine-richdomain of CD6 with 1.4 a 1 : l stoichiometry. J B i d Chem 271:17390-17396. Bowen MA, Bajorath J, D’Egidio M, Whitney GS, Palmer D, Kobarg J, Starling 1.2” GC,Siadak AW, AruffoA.1997.Characterization of mouseALCAM (CD166): The CD6 binding site is conserved in different homologues and 1” mediates cross-species binding. Eur J Immunol 271469-1478. (D Bowcn MA, Patel DD, Li X, Modrell B, Malacko AR, Wang W-C, Marquardt H, Neubauer M,Pesando JM, Francke U, Haynes BF, Aruffo A. 1995. 0.8 Cloning, mapping, and characterization of activated leukocytecell adhesion VI molecule, a CD6 ligand. J Exp Med 181:2213-2220. 0.6 Bums FR, von Kannen S, Guy L, Raper A, Kamholz J, Chang S. 1991. DMGRASP, a novel immunoglobulin superfamily axonal surface protem that 0.4 supports neurite extension. Neuron 7209-220. Gangemi RM, Swack JA, Gaviria DM, Romain PL. 1989. Anti-T12. an anti0.2 CD6 monoclonal antibody, can activate human T lymphocytes. J Immunol 143:2439-2447. Mayer B, Funke I, Seed B, Riethmiiller G, Weiss E. 1990. Expression of the CD6 T lymphocyte differentiation antigen in normal human brain. J Neuro0.1 I IO 100 1000 10000 immunol 29193-202. Morimoto C. Rudd CE, Letvin NL, Hagan M. Schlossman SF. 1988. 2H1-A PROTEIN (nglml) novel antigen involved in T lymphocyte triggering. J Immunol 140:21652 170. Fig. 3. Binding of human ALCAM and BEN Ig fusion proteins to immoPatel DD, Wee S-F, Whichard LP, Bowen MA, Pesando JM, Aruffo A, Haynes bilized full-length CD6-Ig by ELISA. A: Comparison of ALCAM D1D2, BF. 1995. Identification and characterization of a 100-kDa ligand for CD6 BEN D1D2, and BEN DI/ALCAM D2. B: Comparison of full-length on human thymic epithelial cells. J Exp Med 181:1563-1568. ALCAM and BEN. Methods: full length CD6-Ig with mouse IgG2a conPourquie 0,Corbel C, Le Caer J-P, Rosier J, Le Douarin NM. 1992. BEN. a stant regions and ALCAM Dl-, DlD2-, and DID2D3-Ig fusion proteins surface glycoprotein of the immunoglobulin superfamily, is expressed in a with human IgGl constant regions were generated by PCR as described variety of developing systems. Proc Nut/ Acad Sci USA 895261-5265. (Bowen et al., 1996). Full length BEN-Ig and BEN DID2-Ig fusion proResnick D, Pearson A, Krieger M. 1994. TheSRCRsuperfamily:Afamily teins were generated by PCR from an embryonic chicken cDNA library. A reminiscent of the Ig superfamily. Trends Biochem Sci 195-8. hybrid BEN DI/ALCAM D2-Ig fusion protein was constructed from inSkonier JE, Bowen MA, Emswiler J, Aruffo A, Bajorath J. 1996. Mutational dividualdomainsbyoverlapextensionPCR.Theconstructswereconanalysis of the CD6 binding site in activated leukocyte cell adhesion molfirmed by cDNA sequencing. The fusion proteins were transiently expressed ecule. Biochemistry 35:14743-14748. in COS cells (Bowen et al., 1996) and purified on a protein A column. To Whitney GS, Starling GC, Bowen MA, Modrell B, Siadak AW, Aruffo A. 1995. testbinding,full-lengthCD6-Igwasimmobilized on plasticandserial The membrane-proximal scavenger receptor cysteine-rich domain of CD6 dilutions of purified ALCAM and BEN fusion proteinsor COS cell culture contains the activated leukocytecell adhesion molecule binding site. J B i d supernatants with determined protein concentrations were added. Binding Chem 270:18187-18190. Williams AF, Barclay AN. 1988. The immunoglobulin superfamily: domains for IgG antiwas detected using an enzyme-conjugated donkey anti-human cell surface recognition. Annu Rev Immunol 6:381-406. body as described (Skonier et al., 1996).

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