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• W2021106843 abstract "Previous studies have suggested that PECAM-1 mediates cellular interactions via both homophilic and heterophilic adhesive mechanisms. Cell surface glycoaminoglycans have been implicated as one of the heterophilic ligands for PECAM-1. To determine whether PECAM-1 is capable of interacting directly with glycosaminoglycans, we examined the adhesive properties of multiple monovalent and multivalent forms of this adhesion molecule. We found that the binding of a bivalent PECAM-1/IgG chimeric protein or multivalent PECAM-1-containing proteoliposomes to multiple different cell lines was 1) strictly dependent upon cell surface expression of PECAM-1 and 2) unaffected by the presence of excess heparin or heparan sulfate. The extracellular domain of PECAM-1 failed to interact specifically with heparin-Sepharose, 3H-labeled heparin, or a heparin-bovine serum albumin conjugate. In addition, an amino acid sequence motif inadvertently created by the juxtaposition of PECAM-1 and IgG sequences within the hinge region of certain PECAM-1/IgG chimeric constructs was found to confer glycosaminoglycan binding properties not normally present within the extracellular domain of the native molecule. Together, these data suggest that the mechanism by which heparin is able to affect PECAM-1-dependent cell-cell adhesion is indirect and occurs via inhibition of events that occur downstream from PECAM-1 engagement. Previous studies have suggested that PECAM-1 mediates cellular interactions via both homophilic and heterophilic adhesive mechanisms. Cell surface glycoaminoglycans have been implicated as one of the heterophilic ligands for PECAM-1. To determine whether PECAM-1 is capable of interacting directly with glycosaminoglycans, we examined the adhesive properties of multiple monovalent and multivalent forms of this adhesion molecule. We found that the binding of a bivalent PECAM-1/IgG chimeric protein or multivalent PECAM-1-containing proteoliposomes to multiple different cell lines was 1) strictly dependent upon cell surface expression of PECAM-1 and 2) unaffected by the presence of excess heparin or heparan sulfate. The extracellular domain of PECAM-1 failed to interact specifically with heparin-Sepharose, 3H-labeled heparin, or a heparin-bovine serum albumin conjugate. In addition, an amino acid sequence motif inadvertently created by the juxtaposition of PECAM-1 and IgG sequences within the hinge region of certain PECAM-1/IgG chimeric constructs was found to confer glycosaminoglycan binding properties not normally present within the extracellular domain of the native molecule. Together, these data suggest that the mechanism by which heparin is able to affect PECAM-1-dependent cell-cell adhesion is indirect and occurs via inhibition of events that occur downstream from PECAM-1 engagement. PECAM-1 (CD31) is a 130-kDa member of the Ig gene superfamily that is constitutively expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T cell subsets. It is also present at relatively high concentration at the cell junctions of all continuous endothelium, both in vivo and in cell culture (for a recent review on the biology of PECAM-1, see Ref.1Newman P.J. J. Clin. Invest. 1997; 99: 3-8Crossref PubMed Scopus (432) Google Scholar). Because of its presence on these vascular cells, PECAM-1 has been implicated in mediating a number of cellular interactions, most notably those that take place between leukocytes and the vessel wall during the process of transendothelial migration (2Bogen S.A. Baldwin H.S. Watkins S.C. Albelda S.M. Abbas A.K. Am. J. Pathol. 1992; 141: 843-854PubMed Google Scholar, 3Muller W.A. Weigl S.A. Deng X. Phillips D.M. J. Exp. Med. 1993; 178: 449-460Crossref PubMed Scopus (984) Google Scholar, 4Vaporciyan A.A. DeLisser H.M. Yan H.-C. Mendiguren I.I. Thom S.R. Jones M.L. Ward P.A. Albelda S.M. Science. 1993; 262: 1580-1582Crossref PubMed Scopus (429) Google Scholar, 5Bogen S. Pak J. Garifallou M. Deng X. Muller W.A. J. Exp. Med. 1994; 179: 1059-1064Crossref PubMed Scopus (281) Google Scholar, 6Gumina R.J. Schultz J.E. Yao Z. Kenny D. Warltier D.C. Newman P.J. Gross G.J. Circulation. 1996; 94: 3327-3333Crossref PubMed Scopus (79) Google Scholar, 7Wakelin M.W. Sanz M.-J. Dewar A. Albelda S.M. Larkin S.W. Boughton-Smith N. Nourshargh S. J. Exp. Med. 1996; 184: 229-239Crossref PubMed Scopus (169) Google Scholar, 8Murohara T. Delyani J.A. Albelda S.M. Lefer A.M. J. Immunol. 1996; 156: 3550-3557PubMed Google Scholar, 9Zocchi M.R. Ferrero E. Leone B.E. Rovere P. Bianchi E. Toninelli E. Pardi R. Eur. J. Immunol. 1996; 26: 759-767Crossref PubMed Scopus (76) Google Scholar) and between adjacent endothelial cells during the process of angiogenesis (10Matsumura T. Wolff K. Petzelbauer P. J. Immunol. 1997; 158: 3408-3416PubMed Google Scholar, 11Sheibani N. Newman P.J. Frazier W.A. Mol. Biol. Cell. 1997; 8: 1329-1341Crossref PubMed Scopus (75) Google Scholar, 12DeLisser H.M. Christofidou-Solomidou M. Strieter R.M. Burdick M.D. Robinson C.S. Wexler R.S. Kerr J.S. Garlanda C. Merwin J.R. Madri J.A. Albelda S.M. Am. J. Pathol. 1997; 151: 671-677PubMed Google Scholar).A number of different cell surface components have been implicated as counterreceptors or cellular targets for PECAM-1. Albeldaet al. (13Albelda S.M. Muller W.A. Buck C.A. Newman P.J. J. Cell. Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (606) Google Scholar) found that PECAM-1 became concentrated at cell-cell borders only if both cells expressed PECAM-1, and they were the first to propose, based on this observation, that PECAM-1-mediated cellular interactions might operate homophilically, i.e. via PECAM-1/PECAM-1 intermolecular contacts. In support of this hypothesis, PECAM-1-containing proteoliposomes were recently shown to be able to self-associate in a concentration-dependent, divalent cation-independent, manner (14Sun Q.-H. DeLisser H.M. Zukowski M.M. Paddock C. Albelda S.M. Newman P.J. J. Biol. Chem. 1996; 271: 11090-11098Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), providing direct experimental support that PECAM-1 is capable of interacting with itself. Homophilic binding activity requires amino-terminal Ig homology domains 1 and 2 (14Sun Q.-H. DeLisser H.M. Zukowski M.M. Paddock C. Albelda S.M. Newman P.J. J. Biol. Chem. 1996; 271: 11090-11098Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Sun J. Williams J. Yan H.-C. Amin K.M. Albelda S.M. DeLisser H.M. J. Biol. Chem. 1996; 271: 18561-18570Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and specific residues within Ig domain 1 that participate in PECAM-1/PECAM-1 interactions have recently been identified by Newtonet al. (16Newton J.P. Buckley C.D. Jones E.Y. Simmons D.L. J. Biol. Chem. 1997; 272: 20555-20563Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The possibility that the amino-terminal region of the extracellular domain is physiologically relevant to PECAM-1-mediated cellular interactions is further supported by the findings that 1) anti-PECAM-1 monoclonal antibodies (mAbs) 1The abbreviations used are: mAb, monoclonal antibody; huPECAM-1, human PECAM-1; srhuPECAM-1, soluble recombinant huPECAM-1; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt solution; HUVEC, human umbilical vein endothelial cell; TSP, thrombospondin; PF4, platelet factor 4; FPLC, fast protein liquid chromatography; BSA, bovine serum albumin. 1The abbreviations used are: mAb, monoclonal antibody; huPECAM-1, human PECAM-1; srhuPECAM-1, soluble recombinant huPECAM-1; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt solution; HUVEC, human umbilical vein endothelial cell; TSP, thrombospondin; PF4, platelet factor 4; FPLC, fast protein liquid chromatography; BSA, bovine serum albumin. that inhibit leukocyte transendothelial migration almost without exception epitope map to Ig domain 1 or 2 (17Liao F. Huynh H.K. Eiroa A. Greene T. Polizzi E. Muller W.A. J. Exp. Med. 1995; 182: 1337-1343Crossref PubMed Scopus (213) Google Scholar, 18Yan H.-C. Pilewski J.M. Zhang Q. DeLisser H.M. Romer L. Albelda S.M. Cell Adhesion Commun. 1995; 3: 45-66Crossref PubMed Scopus (61) Google Scholar) and 2) soluble Ig domain 1 of PECAM-1 is sufficient to block transendothelial migration in vitroand in vivo (19Liao F. Ali J. Greene T. Muller W.A. J. Exp. Med. 1997; 185: 1349-1357Crossref PubMed Scopus (170) Google Scholar).A number of experimental observations have suggested that PECAM-1 may also be capable of interacting heterophilically with other components of the cell surface. Albelda et al. found that L cell fibroblasts transfected with recombinant PECAM-1 acquired the ability to aggregate with one another in a calcium-dependent manner and raised the possibility that cation-dependent, PECAM-1-mediated cellular interactions might involve additional ligands, such as integrins or proteoglycans (13Albelda S.M. Muller W.A. Buck C.A. Newman P.J. J. Cell. Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (606) Google Scholar). In this regard, these authors noted that PECAM-1 contains within Ig domain 2 the amino acid sequence LKREKN, which corresponds loosely to one of several possible consensus glycosaminoglycan recognition motifs (20Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Shortly thereafter, PECAM-1-transfected L cells were found to bind as readily to nontransfected L cells as they did to each other (21Muller W.A. Berman M.E. Newman P.J. DeLisser H.M. Albelda S.M. J. Exp. Med. 1992; 175: 1401-1404Crossref PubMed Scopus (139) Google Scholar), and the aggregation of PECAM-1-transfected L cells was shown to be inhibitable by selected glycosaminoglycans, including chondroitin 6-sulfate, heparan sulfate, and heparin, as well as by the Ig domain 2-derived synthetic peptide, LKREKN (22DeLisser H.M. Yan H.-C.Y. Newman P.J. Muller W.A. Buck C.A. Albelda S.M. J. Biol. Chem. 1993; 268: 16037-16046Abstract Full Text PDF PubMed Google Scholar). Taken together, these data have led to the widely held supposition that PECAM-1 is a heparin-binding protein.Given the potential importance of PECAM-1 in regulating the interaction of vascular cells during the processes of inflammation, thrombosis, and angiogenesis, determining the range of potential cellular targets for PECAM-1 is crucial for understanding the mechanism by which it is able to both initiate and respond to adhesive and signaling events. The purpose of the present investigation, therefore, was to specifically determine whether PECAM-1 is capable of interacting directly with glycosaminoglycans. To accomplish this aim, we have examined the adhesive properties of multiple mono- and multivalent forms of this adhesion receptor, including full-length cellular PECAM-1 derived from human platelet membranes, monomeric soluble recombinant human PECAM-1 (srhuPECAM-1), PECAM-1-containing proteoliposomes, and a bivalent recombinant huPECAM-1/IgG chimeric protein.DISCUSSIONAlthough PECAM-1 was discovered as a novel membrane glycoprotein on the surface of platelets, leukocytes, and endothelial cells more than 10 years ago (28van Mourik J.A. Leeksma O.C. Reinders J.H. de Groot P.G. Zandbergen-Spaargaren J. J. Biol. Chem. 1985; 260: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 29Ohto H. Maeda H. Shibata Y. Chen R.-F. Qzaki Y. Higashihara M. Takeuchi A. Tohyama H. Blood. 1985; 66: 873-881Crossref PubMed Google Scholar, 30Newman P.J. Doers M.P. Gorski J. J. Cell. Biol. 1987; 105 (abstr.): 53aCrossref Scopus (68) Google Scholar), its precise role in the biology of blood and vascular cells is still being defined. Recent studies have established a role for PECAM-1 in leukocyte transendothelial migration (2Bogen S.A. Baldwin H.S. Watkins S.C. Albelda S.M. Abbas A.K. Am. J. Pathol. 1992; 141: 843-854PubMed Google Scholar, 3Muller W.A. Weigl S.A. Deng X. Phillips D.M. J. Exp. Med. 1993; 178: 449-460Crossref PubMed Scopus (984) Google Scholar, 4Vaporciyan A.A. DeLisser H.M. Yan H.-C. Mendiguren I.I. Thom S.R. Jones M.L. Ward P.A. Albelda S.M. Science. 1993; 262: 1580-1582Crossref PubMed Scopus (429) Google Scholar, 5Bogen S. Pak J. Garifallou M. Deng X. Muller W.A. J. Exp. Med. 1994; 179: 1059-1064Crossref PubMed Scopus (281) Google Scholar, 6Gumina R.J. Schultz J.E. Yao Z. Kenny D. Warltier D.C. Newman P.J. Gross G.J. Circulation. 1996; 94: 3327-3333Crossref PubMed Scopus (79) Google Scholar, 7Wakelin M.W. Sanz M.-J. Dewar A. Albelda S.M. Larkin S.W. Boughton-Smith N. Nourshargh S. J. Exp. Med. 1996; 184: 229-239Crossref PubMed Scopus (169) Google Scholar, 8Murohara T. Delyani J.A. Albelda S.M. Lefer A.M. J. Immunol. 1996; 156: 3550-3557PubMed Google Scholar, 9Zocchi M.R. Ferrero E. Leone B.E. Rovere P. Bianchi E. Toninelli E. Pardi R. Eur. J. Immunol. 1996; 26: 759-767Crossref PubMed Scopus (76) Google Scholar), in up-regulating integrin function (31Tanaka Y. Albelda S.M. Horgan K.J. Van Seventer G.A. Shimizu Y. Newman W. Hallam J. Newman P.J. Buck C.A. Shaw S. J. Exp. Med. 1992; 176: 245-253Crossref PubMed Scopus (338) Google Scholar, 32Piali L. Albelda S.M. Baldwin H.S. Hammel P. Gisler R.H. Imhof B.A. Eur. J. Immunol. 1993; 23: 2464-2471Crossref PubMed Scopus (120) Google Scholar, 33Leavesley D.I. Oliver J.M. Swart B.W. Berndt M.C. Haylock D.N. Simmons P.J. J. Immunol. 1994; 153: 4673-4683PubMed Google Scholar, 34Berman M.E. Muller W.A. J. Immunol. 1995; 154: 299-307PubMed Google Scholar, 35Berman M.E. Xie Y. Muller W.A. J. Immunol. 1996; 156: 1515-1524PubMed Google Scholar, 36Varon D. Jackson D.E. Shenkman B. Dardik R. Tamarin I. Savion N. Newman P.J. Blood. 1998; 91: 500-507Crossref PubMed Google Scholar), and in angiogenesis (10Matsumura T. Wolff K. Petzelbauer P. J. Immunol. 1997; 158: 3408-3416PubMed Google Scholar, 11Sheibani N. Newman P.J. Frazier W.A. Mol. Biol. Cell. 1997; 8: 1329-1341Crossref PubMed Scopus (75) Google Scholar, 12DeLisser H.M. Christofidou-Solomidou M. Strieter R.M. Burdick M.D. Robinson C.S. Wexler R.S. Kerr J.S. Garlanda C. Merwin J.R. Madri J.A. Albelda S.M. Am. J. Pathol. 1997; 151: 671-677PubMed Google Scholar). Moreover, it has recently become apparent that, following cellular activation or engagement, the cytoplasmic domain of PECAM-1 may become tyrosine-phosphorylated (37Jackson D.E. Ward C.M. Wang R. Newman P.J. Blood. 1996; 88 (abstr.): 438aGoogle Scholar, 38Levine B.L. Ueda Y. Faith A. June C.H. Tissue Antigens. 1996; 48: 319-324Crossref PubMed Scopus (5) Google Scholar, 39Lu T.T. Yan L.G. Madri J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11808-11813Crossref PubMed Scopus (97) Google Scholar, 40Sagawa K. Swaim W. Zhang J. Unsworth E. Siraganian R.P. J. Biol. Chem. 1997; 272: 13412-13418Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 41Osawa M. Masuda M. Harada N. Lopes R.B. Fujiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar), serving as a docking site for one or more cytosolic signaling molecules (42Jackson D.E. Ward C.M. Wang R. Newman P.J. J. Biol. Chem. 1997; 272: 6986-6993Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 43Masuda M. Osawa M. Shigematsu H. Harada N. Fujiwara K. FEBS Lett. 1997; 408: 331-336Crossref PubMed Scopus (91) Google Scholar, 44Jackson D.E. Kupcho K.R. Newman P.J. J. Biol. Chem. 1997; 272: 24868-24875Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 45Lu T.T. Barreuther M. Davis S. Madri J.A. J. Biol. Chem. 1997; 272: 14442-14446Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In an effort to understand the molecular mechanisms underlying these events, we and others have been investigating the adhesive properties of PECAM-1 and have found that amino-terminal Ig homology domains 1 and 2 of PECAM-1 play a key role in mediating PECAM-1/PECAM-1 homophilic interactions (14Sun Q.-H. DeLisser H.M. Zukowski M.M. Paddock C. Albelda S.M. Newman P.J. J. Biol. Chem. 1996; 271: 11090-11098Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Sun J. Williams J. Yan H.-C. Amin K.M. Albelda S.M. DeLisser H.M. J. Biol. Chem. 1996; 271: 18561-18570Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Newton J.P. Buckley C.D. Jones E.Y. Simmons D.L. J. Biol. Chem. 1997; 272: 20555-20563Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Liao F. Huynh H.K. Eiroa A. Greene T. Polizzi E. Muller W.A. J. Exp. Med. 1995; 182: 1337-1343Crossref PubMed Scopus (213) Google Scholar, 19Liao F. Ali J. Greene T. Muller W.A. J. Exp. Med. 1997; 185: 1349-1357Crossref PubMed Scopus (170) Google Scholar). In addition to PECAM-1 itself, however, a number of other cell surface components, including glycosaminoglycans (13Albelda S.M. Muller W.A. Buck C.A. Newman P.J. J. Cell. Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (606) Google Scholar, 21Muller W.A. Berman M.E. Newman P.J. DeLisser H.M. Albelda S.M. J. Exp. Med. 1992; 175: 1401-1404Crossref PubMed Scopus (139) Google Scholar, 22DeLisser H.M. Yan H.-C.Y. Newman P.J. Muller W.A. Buck C.A. Albelda S.M. J. Biol. Chem. 1993; 268: 16037-16046Abstract Full Text PDF PubMed Google Scholar, 46Watt S.M. Williamson J. Genevier H. Fawcett J. Simmons D.L. Harzfield A. Nesbitt S.A. Coombe D.R. Blood. 1993; 82: 2649-2663Crossref PubMed Google Scholar) have been implicated as ligands for PECAM-1. The purpose of the present study, therefore, was to determine whether the extracellular domain of PECAM-1 contains one or more functional glycosaminoglycan-binding sites and whether cell surface glycosaminoglycans can serve as a biologically relevant target for PECAM-1-mediated cellular interactions.To accomplish these aims, we examined the glycosaminoglycan binding properties of multiple mono- and multivalent forms of PECAM-1. Our results may be summarized as follows. First, the interaction with cells of an appropriately constructed bivalent PECAM-1/IgG chimeric protein is strictly dependent on cell surface expression of PECAM-1 (Fig. 1), and exogenously added glycosaminoglycans such as heparin and heparan sulfate have no effect on the ability of PECAM-1/IgG or PECAM-1 proteoliposomes to interact with the cell surface (Fig. 2 A). Second, even in cells rich in cell surface glycosaminoglycans and integrins, PECAM-1 appears to be the only functional cellular target for PECAM-1, since the interaction of PECAM-1/IgG with the endothelial cell surface could be completely inhibited by preincubating the cells with small Fab fragments specific for PECAM-1 Ig homology domain 1 (Fig. 2 B). Third, independent of either the source of PECAM-1 or the way in which heparin is presented, the extracellular domain of PECAM-1 is unable to interact directly with this glycosaminoglycan (Figs. 3 and 4, and Table I). Finally, we found that amino acid sequences contributed by certain Ig fusion vectors can, in some instances, result in the creation of artifactual heparin-binding sites that are normally not present in the native molecule (Fig. 5).Several other groups have examined the association of heparin with various cellular and chimeric forms of PECAM-1 and arrived at somewhat different conclusions from those drawn in the present work. Wattet al. (46Watt S.M. Williamson J. Genevier H. Fawcett J. Simmons D.L. Harzfield A. Nesbitt S.A. Coombe D.R. Blood. 1993; 82: 2649-2663Crossref PubMed Google Scholar) examined the retention on heparin-Sepharose beads of full-length PECAM-1 derived from detergent cell lysates and found that approximately 20% of the PECAM-1 remained bound to the beads after washing with normal saline. However, even the small proportion of PECAM-1 that remained bound to the heparin-Sepharose beads was found to be easily dissociable. Based upon their observations, they concluded that PECAM-1 exhibited weak, but measurable, low affinity interactions with heparin. To more specifically localize the site on full-length PECAM-1 responsible for this weak association, we compared the heparin binding properties of srhuPECAM-1, a recombinant protein containing only amino acid residues 1–574 (i.e. the entire extracellular domain of PECAM-1 containing Ig-homology domains 1–6), with that of full-length PECAM-1 derived from human platelet membranes. In physiological saline, srhuPECAM-1 exhibited no affinity for heparin immobilized on Sepharose beads (Fig. 3), for heparin in solution (Fig. 4), or for heparin conjugated to BSA (Table I). However, similar to Watt et al., we did observe a weak interaction of heparin withfull-length PECAM-1 purified from cellular detergent lysates (Table I). These data suggest that the heparin-binding site on PECAM-1 lies not within the extracellular domain but rather within either the transmembrane or cytoplasmic domain of the molecule. In this regard, it is notable that there are a series of positively charged, basic amino acids immediately following the transmembrane domain of PECAM-1, having the sequence RKAKAK (residues 599–604), that confer weak heparin binding properties to the protein independent of those that might be present within the extracellular domain. In light of these findings, we suspect that the ability of full-length cell-derived PECAM-1 to bind to heparin-Sepharose (Ref. 46Watt S.M. Williamson J. Genevier H. Fawcett J. Simmons D.L. Harzfield A. Nesbitt S.A. Coombe D.R. Blood. 1993; 82: 2649-2663Crossref PubMed Google Scholar and Table I) is probably due to the association of cytoplasmic domain residues 599–604 with heparin. This interaction is likely to be of little or no functional consequence in PECAM-1-mediated cellular interactions, since heparin-binding sequences not present in the extracellular domain of the molecule would obviously be unavailable to mediate heterophilic interactions between cells.An important aspect of the present work is the finding that short amino acid sequences encoded by certain PECAM-1/IgG cDNA constructs, but not by others, can confer artifactual heparin binding properties to the chimeric protein. Margalit et al. (47Margalit H. Fischer N. Ben-Sasson S.A. J. Biol. Chem. 1993; 268: 19228-19231Abstract Full Text PDF PubMed Google Scholar) have recently shown, using three-dimensional computer graphic techniques, that heparin binding sites within proteins are dependent upon electrostatic interactions that result from topologically close basic amino acid residues. Thus, PECAM-1/IgGRR, but not PECAM-1/IgG, contains the sequence KKAAARR within its hinge region (Fig. 5 A), and this motif is by itself able to contribute significantly to the glycosaminoglycan-binding ability of proteins and polypeptides that contain it (Figs. 5, B andC). In fact, PECAM-1/IgGRR binds a variety of PECAM-1-positive and PECAM-1-negative cell lines in a heparin-inhibitable manner. 2Q-H. Sun and P. J. Newman, unpublished observations. By themselves, these observations would have led us to believe that PECAM-1 is a heparin-binding protein and that cell surface glycosaminoglycans represent a functional target for PECAM-1 on the cell surface. However, our discovery that simple deletion of theKKAAARR motif totally eliminates the ability of PECAM-1/IgG to bind to PECAM-1-negative cells argues strongly against this possibility. Thus, we speculate that the 6–30% observed binding to heparin of the particular PECAM-1/IgG construct used by Wattet al. (Fig. 8 of Ref. 46Watt S.M. Williamson J. Genevier H. Fawcett J. Simmons D.L. Harzfield A. Nesbitt S.A. Coombe D.R. Blood. 1993; 82: 2649-2663Crossref PubMed Google Scholar) may similarly be attributable, at least in part, to sequence motifs unknowingly introduced into the hinge region of their chimeric protein that are normally notpresent within in the extracellular domain of cellular PECAM-1. The presence of similar artificially juxtaposed amino acid residues might also explain the findings of Prager et al. (48Prager E. Sunder-Plassmann R. Hansmann C. Koch C. Holter W. Knapp W. Stockinger H. J. Exp. Med. 1996; 184: 41-50Crossref PubMed Scopus (70) Google Scholar), who observed heparin-inhibitable binding to T cells of their form of PECAM-1/IgG.The artifactual binding of PECAM-1/IgGRR to cells, independent of their expression of the natural ligand for PECAM-1, may not be limited to PECAM-1-based immunoadhesins. There appears, in the immunoadhesin literature in general, to have been little consideration given to the possibility that the adhesive properties of IgG fusion proteins may be affected by a combination of sequence motifs, some of which are not normally present in the native molecule. For example, in addition to sequences that may be unknowingly introduced into the hinge regions of chimeric proteins, many immunoadhesin cDNA expression vectors encode a polyhistidine tag at the carboxyl terminus to simplify recovery and purification. Unfortunately, clusters of positively charged histidine residues are also able to contribute to the overall affinity of proteins for cell surface glycosaminoglycans (49Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Thus, while hinge region sequences or polyhistidine tags by themselves may be unable to confer high affinity heparin binding characteristics, when expressed together with low affinity sites present within the native protein, they may be able to significantly alter the overall cell binding properties of the chimeric protein.Previous results from our laboratory and others have led to the hypothesis that the six-amino acid sequence, LKREKN, located at amino acid residues 150–155 within extracellular Ig domain 2, might function as a heparin binding site (13Albelda S.M. Muller W.A. Buck C.A. Newman P.J. J. Cell. Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (606) Google Scholar). This hypothesis was based 1) on the similarity of this sequence with a known glycosaminoglycan binding motif having the sequenceX −3-B−2-B−1-X +1-B+2-X +3, where B is a basic amino acid and X is hydropathic residue (20Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) and 2) on the ability of both heparin and the LKREKN peptide to inhibit the in vitro association of PECAM-1-transfected L cells with nontransfected L cells (22DeLisser H.M. Yan H.-C.Y. Newman P.J. Muller W.A. Buck C.A. Albelda S.M. J. Biol. Chem. 1993; 268: 16037-16046Abstract Full Text PDF PubMed Google Scholar). There are, however, a number of theoretical problems and recent observations that cast doubt on the ability of LKREKN to serve as a functional heparin binding domain. First, the reverse peptide, NKERKL, which does not contain theXBBXBX motif, was found to be equally inhibitory in the L cell aggregation assay, and neither the forward nor the reverse peptide exhibited significant inhibitory effects except at a relatively high concentration (>0.5 mm) (22DeLisser H.M. Yan H.-C.Y. Newman P.J. Muller W.A. Buck C.A. Albelda S.M. J. Biol. Chem. 1993; 268: 16037-16046Abstract Full Text PDF PubMed Google Scholar). Second, as shown in Table II, it has only recently become appreciated that neither the LKREKN sequence nor theXBBXBX motif are particularly well conserved evolutionarily in PECAM-1; in rat PECAM-1, the −2-position is occupied by a serine rather than the obligatory Lys or Arg residue, and the basic residue normally found at −1 is occupied in murine PECAM-1 by an isoleucine. In fact, a perfectly conserved glutamic acid residue occupies the normally hydropathic +1-position in all PECAM-1 species examined to date, and negatively charged residues are specifically excluded from functional glycosaminoglycan-binding motifs because they have the potential to significantly alter the charge complementation of this region necessary to effect its interaction with negatively charged heparin (20Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Third, Piali et al. (50Piali L. Hammel P. Uherek C. Bachmann F. Gisler R.H. Dunon D. Imhof B.A. J. Cell. Biol. 1995; 130: 451-460Crossref PubMed Scopus (340) Google Scholar) have recently found that the adhesion of murine lymphokine-activated killer cells to an immobilized chimeric protein composed of murine PECAM-1 Ig domains 1–3 fused to the mouse Igκ constant region could be inhibited by anti-PECAM-1 antibodies but wasnot inhibitable by heparin at concentrations as high as 300 μg/ml (50Piali L. Hammel P. Uherek C. Bachmann F. Gisler R.H. Dunon D. Imhof B.A. J. Cell. Biol. 1995; 130: 451-460Crossref PubMed Scopus (340) Google Scholar). Fourth, neither heparin (100 μg/ml) nor the LKREKN peptide (1 mg/ml) had any effect on the adhesion of U937 cells to immobilized full-length PECAM-1/IgG (51Buckley C.D. Doyonnas R. Newton J.P. Blystone S.D. Brown E.J. Watt S.M. Simmons D.L. J. Cell Sci. 1996; 109: 437-445PubMed Google Scholar). Finally, neither leukocyte transendothelial migration nor endothelial cell tube formation, each of which are thought to involve PECAM-1, are inhibited by heparin (10Matsumura T. Wolff K. Petzelbauer P. J. Immunol. 1997; 158: 3408-3416PubMed Google Scholar,17Liao F. Huynh H.K. Eiroa A. Greene T. Polizzi E. Muller W.A. J. Exp. Med. 1995; 182: 1337-1343Crossref PubMed Scopus (213) Google Scholar). Taken together with the fact that heparin has no affinity for synthetic peptides containing the LKREKN sequence,2 these data suggest that the mechanism by which heparin is able to inhibit the interaction of PECAM-1-transfected L cells would appear to be independent of the direct glycosaminoglycan binding properties of PECAM-1 itself and probably operates by inhibiting secondary adhesive interactions that occur downstream of PECAM-1 (1Newman P.J. J. Clin. Invest. 1997; 99: 3-8Crossref PubMed Scopus (432) Google Scholar). Identification of the range of cellular and molecular events that emanate from PECAM-1 engagement remains an impo" @default.
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• W2021106843 cites W1537851214 @default.
• W2021106843 cites W1544261227 @default.
• W2021106843 cites W1549949535 @default.
• W2021106843 cites W1563596243 @default.
• W2021106843 cites W1583356042 @default.
• W2021106843 cites W1596750278 @default.
• W2021106843 cites W1603416910 @default.
• W2021106843 cites W1833499979 @default.
• W2021106843 cites W1873722097 @default.
• W2021106843 cites W1932316892 @default.
• W2021106843 cites W1964570763 @default.
• W2021106843 cites W1967956055 @default.
• W2021106843 cites W1969930542 @default.
• W2021106843 cites W1970746962 @default.
• W2021106843 cites W1975651989 @default.
• W2021106843 cites W1976658012 @default.
• W2021106843 cites W1982687474 @default.
• W2021106843 cites W1986070215 @default.
• W2021106843 cites W2015184074 @default.
• W2021106843 cites W2025142983 @default.
• W2021106843 cites W2033546665 @default.
• W2021106843 cites W2049760106 @default.
• W2021106843 cites W2049866297 @default.
• W2021106843 cites W2051889011 @default.
• W2021106843 cites W2055970438 @default.
• W2021106843 cites W2058688854 @default.
• W2021106843 cites W2060176983 @default.
• W2021106843 cites W2076544464 @default.
• W2021106843 cites W2084830265 @default.
• W2021106843 cites W2085436260 @default.
• W2021106843 cites W2091002608 @default.
• W2021106843 cites W2092751827 @default.
• W2021106843 cites W2101688560 @default.
• W2021106843 cites W2109342412 @default.
• W2021106843 cites W2116133156 @default.
• W2021106843 cites W2117538259 @default.
• W2021106843 cites W2157258908 @default.
• W2021106843 cites W2159265736 @default.
• W2021106843 cites W2166833739 @default.
• W2021106843 cites W2169429927 @default.
• W2021106843 cites W2169778726 @default.
• W2021106843 cites W2247620787 @default.
• W2021106843 cites W2347114980 @default.
• W2021106843 cites W2417318333 @default.
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