ILL Glycobiology vol. 2 no 5 pp. 395^00. 1992
1 5. OCT
Glyco-Forum section Letter to the Glyco-Forum Evidence for two classes of carbohydrate binding sites on selectins Darwin Asa, Tracey Gant, Yuko Oda and Brian K.Brandley Glycomed, Inc., 860 Atlantic Avenue, Alameda. CA 94501, USA
Sialyl Lewis x ligands The ability of the selectins to bind to sialyl Lewis x oligosaccharides has been previously reported by a number of groups, using several different techniques. There is now general agreement that E-Selectin recognizes NeuNAc a2-3 Gal/31-4 (Fuccd-3) GlcNAc (sialyl Lewis x, or sLex) and related oligosaccharides (Lowe et al., 1990; Phillips et al., 1990; Walz et al., 1990; Berg et al., 1991; Tiemeyer et al., 1991; Tyrrell et al., 1991). P-Selectin has been reported to bind to the Lewis x structure (Gal /31-4 (Fucal-3) GlcNAc) (Larsen et al., 1990) and/or sLe" (Polley et al., 1991; Foxall et al., 1992). Recently we reported that L-Selectin can also recognize the sLe* structure (Foxall et al., 1992; Figure 1A). Selectin binding to sLe" is dependent on divalent cations, but not exclusively calcium (Figure 2). Strontium is capable of replacing calcium to restore binding of E-Selectin to sLex. For L-Selectin, copper, in addition to strontium, can substitute for calcium. When using P-Selectin, strontium and magnesium (partially) can restore binding to sLe". This effect of magnesium on P-Selectin binding to synthetic sLex is consistent with the observations of Geng et al. (1990) who reported that magnesium could stimulate P-Selectin-mediated neutrophil adhesion at suboptimal levels of calcium. Oxford University Press
Carbohydrates as dissimilar as yeast phosphomannan (PPME), an algal polysaccharide containing fucose sulphate (fucoidan) and sulphatides were used as tools to examine L-Selectin binding activity (Stoolman and Rosen, 1983; Imai et al., 1990), even before the selectins were recognized as a class of related proteins. In addition to sialyl Lewis x, all three selectins bind to sulphatides [Figure IB and Imai et al. (1990)], and L- and P-Selectins bind to SulphoGlucuronylNeoLacto (SGNL) lipid, an HNK-1 reactive epitope (Figure 1C). The direct binding of L- and P-Selectins to SGNL lipid is the clearest demonstration of the similarity of these two receptors, as well as their functional separation from E-Selectin. All the carbohydrate structures recognized by the selectins possess negative charge, but despite this they are very structurally dissimilar. The specificity of these interactions is indicated by three lines of evidence, (i) The oligosaccharide charge alone is not sufficient for recognition since many negatively charged structures (both carbohydrate and non-carbohydrate in nature) are not recognized, (ii) Antibodies known to block selectin function can inhibit the binding of the appropriate selectin to each of the recognized carbohydrate structures (unpublished observations), (iii) Proteins such as human IgG and CD4—IgG chimera do not bind to these carbohydrates, controlling for any artifactual binding of the chimera constructs. The presence of a sulphate ester and uronic acid suggests a similarity between SGNL lipid and glycosaminoglycans (GAGs). GAGs containing primarily iduronic acids (heparin and dermatan sulphate) inhibit L- and P-Selectin binding to sLex and sulphatides (Table I). Other GAGs (chondroitin sulphate, keratan sulphate and Escherichia coli K5) are less potent inhibitors or are ineffective at inhibiting selectin binding. Selectin binding to SGNL appears to be less sensitive to inhibition by GAGs (unpublished observations), but heparin and dermatan sulphate were still more effective than chondroitin sulphate, keratan sulphate or K5. In addition, L- and P-Selectin bind to heparin adsorbed to microtitre plates, or immobilized on agarose beads, in a calcium-independent manner (unpublished observations). E-Selectin is not inhibited by any GAG tested and does not bind to heparin affinity columns (unpublished observations). There are conflicting reports in the literature as to the ability of GAGs to inhibit selectin binding. Rosen's group reported the ability of heparin to inhibit L-Selectin activity in vitro at 20-100 /tg/ml (Stoolman and Rosen, 1983; Imai et al., 1990), but heparin was inactive in other studies when used at 25 jtg/ml. Several laboratories have used heparin at from 100 /tg/ml to 5 mg/ml to block P-Selectin activity, but data from only a single heparin concentration are reported (Aruffo et al., 1991; Handa et al., 1991; Skinner et al., 1991). Skinner et al. (1989) describe the ability of P-Selectin to bind to a heparin affinity column. They also report that the interaction of heparin with P-Selectin is calcium independent (see above). Our data indicate that at least under certain assay conditions, heparin can block L- and P-Selectin function at relatively low concentrations.
395
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The selectins (LEC-CAMs) are a family of three homologous receptors implicated in the initial interactions between leukocytes and vascular endothelia leading to lymphocyte homing, platelet binding and neutrophil extravasation (Luscinskas et al., 1989; Hallman et al., 1991; Lawrence and Springer, 1991; Watson et al., 1991). E-Selectin (LECAM-2, ELAM-1), L-Selectin (LECAM-1, LAM-1, gp90MEL) and P-Selectin (LECAM-3, GMT-140, PADGEM) each contain a domain with homology to calcium-dependent lectins (Bevilacqua et al., 1989; Johnston et al., 1989; Lasky et al., 1989; Tedder et al., 1989), which has led to an intense effort to define carbohydrate ligands for these proteins. Sialyl Lewis x. (NeuNAc a2-3 Gal/31-4 (Fuc a 1-3) GlcNAc) has been identified as a potential ligand for all three selectins (Figure 1A), but this simple onereceptonone-ligand interaction may not explain the complex biology of the receptors. While differential expression of receptors or ligands may account for the observed biological specificity, we feel that there is significant evidence suggesting that multiple carbohydrate binding sites exist on the selectins.
Carbohydrate ligands unrelated to sialyl Lewis x
Glyco-Forum section
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Fig. 1. Selectin adhesion to glycolipids. The adhesion of selectms to adsorbed glycolipid was performed by ELISA as described in Foxall el al. (1992). Briefly, 25 pmol/well glycolipid were adsorbed to microtitre wells overnight. The plates were rinsed and then blocked with 5% bovine serum albumin solution for 1 h. After blocking, plates were rinsed and selectin—IgG chimera, biotinylated anti-human IgG and Streptavidin-AP were added to glycolipid-coated plates and incubated for 45 mm at 37 °C. After binding, the plate was rinsed and binding read using AP substrate at 405 nm. (A) Adhesion of selectins to 2,3 sLe* glycolipid. (B) Adhesion of selectins to sulphatides. (C) Adhesion of selectins to SGNL lipid. Closed symbols in A and B represent adhesion to 2,6 sLe* glycolipid.
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Fig. 2. Effect of Ca replacement by other divalent cations on selectin binding. Buffers used in the ELISA assay as described in Foxall el al. (1992) were pretreated to remove divalent cations by passing them over a Chelex 100 (Bio-Rad) column. After cation depletion, divalent cations (at concentrations from left to right of 2.5. 1.25 and 0.625 mM) were added back to cation-depleted buffers and the ability of those cations to support selectin binding to 2.3 sLe* glycolipid was assessed using the previously described ELISA procedure. (A) E-Selectin; (B) L-Selectin: (C) P-Selectin.
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Glyco-Forum section
Table I. Inhibition of selectin binding by GAGs. The ability of several GAGs to inhibit the binding of selectins was assayed using the previously described EL1SA. GAGs were incubated with the selectin complex for 45 min prior to the addition of selectin to glycolipid-coated wells. Only data on L- and P-Selectin are presented as GAGs do not inhibit E-Selectm binding L-Selectin Surface (% of control binding) GAG Heparin
[GAG] (Mg/ml) 10 1 01 0.01
2,3 sLe*
Sulphatides
25 2 ± 2.5
37.5 ± 3.7
56.1 ± 3.4 81 9 ± 2 8 88 ± 2.6
70.3 ± 1.2 93.1 ± 1.8 91.8 ± 6.1
92.8 76 6 87 2 90.1
+ 7.8 ± 2.2 ± 7.3 ±9
111 6 105.2 102 5 123 7
± ± ± ±
Dermatan sulphate
100 10 1 0.1
23 7 46.4 103 112.6
± 2.5 ±49 ± 9.8 ± 9.1
45.3 87.2 110.3 115.1
±39 ±38 ±47 ± 3.1
K5 (E.coh)
100 10 1 0.1
101.3 101 106 105.6
±42 ± 2 ± 1.2 ±-113
1147 102.1 102 8 121 1
± ± ± ±
12.7 4.8 52 9.2
Keratan sulphate
250 125 62.5 31.25
115.7 127 5 104.3 106.3
± ± ± ±
94.2 107.2 112.6 98.9
± ± ± ±
9.1 10.5 4.1 3.5
3.1 5.3 7.3 5.1
15.6 5.8 0.8 5.5
P-Selectin Surface (% of control binding) GAG Heparin
[GAG] (/ig/ml)
2.3 sLe"
Sulphatides
10 1 01 001
8.6 10.3 24.1 100
± + ± ±
1.1 1.5 2.3 5.9
20.6 19.1 42 3 89.7
± ± ± ±
0.7 0.8 3.4 4.2
250 125 62.5 31.25
14.1 25.9 33.9 55.5
±05 ± 4.6 ± 2.1 ± 5.3
32.9 40 67.9 73.9
± ± ± ±
1.3 1 8.3 1.4
Dermatan sulphate
100 10 1 0.1
8.7 9.6 17.6 92.6
± ± + ±
0.7 0.4 0.2 5.9
12.7 15.3 28.6 79
± + ± ±
0.7 1.4 0.2 4.2
K5 (E.coli)
100 10 1 0.1
76.3 116.7 110.4 106.8
± ± ± ±
0.7 5.1 6.2 4.6
76 84.3 102.3 89 3
± ± ± ±
1.6 2.2 3.9 8.5
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250 125 62.5 31.25
90.9 89.7 876 93.5
± ± ± ±
5.1 3.6 6 2.2
95.9 107.3 117.3 103.7
± ± ± ±
5 5.2 7.5 4.5
Chond sulphate
The binding of all selectins to sLe\ and E-Selectin binding to sulphatides, is calcium dependent. However, the binding of L- and P-Selectins to sulphatides and SGNL lipid is only partially blocked by the removal of calcium and the addition of as little as 100 nM EDTA. The addition of increasing amounts of EDTA shows little further reduction up to 5 mM EDTA (Figure 3). This biphasic response to EDTA suggests two modes of interaction of these selectins with sulphatides and SGNL lipid: one calcium dependent and one calcium independent. L-Selectin binding to fucoidan, and L- and P-Selectin binding to heparin, are also largely calcium-independent recognition events (unpublished data).
Specificity Since the selectins seem to recognize several oligosaccharides by what may be multiple binding sites, one may reasonably question how to define specific carbohydrate binding. By the narrowest definition, specific recognition may require calciumdependent binding of structurally related oligosaccharides to one site in the C-lectin domain. However, many mammalian lectins are known that do not require divalent cations for carbohydrate binding (Drickamer, 1988). Furthermore, many proteins not normally considered lectins do bind carbohydrates that include heparin and sulphatides. Several proteins without identified lectin domains have been reported to bind to sulphatides (Roberts et ai, 1986), including laminin, von Willebrand factor (vWF) and thrombospondin. Others have been reported to bind to SGNL epitope (Loveless et al., 1992) and heparin (Burgess and Maciag, 1989). Such calciumindependent carbohydrate recognition has been considered specific because relatively well-defined structural elements of the carbohydrates were required. By this broader definition, sulphatide and SGNL lipid binding to selectins may be considered specific, even though it is calcium independent. So there is precedent to suggest that portions of the selectins, perhaps outside of the C-lectin domain (i.e. EGF or CBP-like domains), may mediate binding to carbohydrates distinct from those recognized by the lectin domain.
Are there two carbohydrate binding sites on L- and P-Selectin? All data currently available indicate that E-Selectin recognizes sLe" and related carbohydrates by a mechanism that is completely calcium dependent. The story for L- and P-Selectins seems more complex. The ability of these selectins to bind to varied carbohydrate structures suggests that more than one binding site may be involved. Several experiments presented here support this view, (i) The binding of L- and P-Selectins to sulphatides, SGNL lipid and heparin exhibits a biphasic response to the addition of EDTA. This is in contrast to selectin binding to sLe", which is completely blocked with very low levels of EDTA. (ii) The pH optimum for binding to sLex is 7, while the optimum for binding to sulphatides is 5 (unpublished observation), again suggesting different sites, (iii) Binding of selectins to sulphatides is relatively insensitive to increased 397
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250 125 62.5 31.25
Chond sulphate
Cation chelation
Glyco-Forum section
that L- and P-Selectins bind to sulphatides and SGNL lipid by a site that is functionally distinct from the sLex binding site. It is unclear whether both sites reside in the lectin domain, or if the calcium-independent binding site resides in the EGF or CBP-like domains. Inhibition of binding to all putative ligands by antibodies and polysaccharides suggests that the two sites interact in some way, either being physically contiguous or overlapping, or by induction of a conformational change at distant sites (Handa et ai, 1991). We feel that the possibility of mutiple sugar binding sites on selectins may help explain their biological activity. Identification of a second carbohydrate binding site on selectins provides another target for the design of carbohydrate-based inhibitors of the selectins.
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ionic strength (up to 500 mM) compared to binding to sLe\ which exhibits optimal binding at 150—200 mM NaCl (unpublished observation), (iv) Cation inhibitors also discriminate between sLex and sulphatide binding. Barium (at 1 mM) can inhibit the binding of all three selectins to sLe\ but it is relatively ineffective at blocking selectin binding to sulphatides (unpublished observations). Taken together, these data suggest 398
Aruffo.A., Kolanus,W., Walz.G.. Fredman,P. and Seed.B. (1991) CD62/ P-Selectin recognition of myeloid and tumor cell sulfatides Cell, 67, 35—44. Berg.E.L . Robinson,M.K., Mansson.O., Butcher,E.C. and Magnani.J.L (1991) A carbohydrate domain common to both sialyl Le" and sialyl Le* is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1 J. Biol. Chem.. 265, 14869-14872. Bevilacqua,M.P., Stengelin,S., Gimbrone,M.A.,Jr and Seed.B. (1989) Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins Science, 243. 1160-1165. Burgess,W.H and Maciag.T (1989) The Heparin-Binding Growth Factor family of proteins. Annu. Rev. Biochem.. 58, 575—606. Drickamer,K. (1988) Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem., 263, 9557-9560. Foxall.C.F., Watson,S.R., Dowbenko.D., Fenme.C, Lasky.L.A., Kiso,M.. Hasegawa.A., Asa,D. and Brandley.B.K. (1992) The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis x oligosaccharide. J. Cell Biol., in press. Geng,J.-G., Moore,K.L., Johnson.A.E. and McEver,R.P. (1991) Neutrophil recognition requires a Ca2+-induced conformational change in the lectin domain of GMP-140. J. Biol. Chem., 266. 22313-22318. Hallman,R , Jutila,M.A., Smith,C.W , Anderson,D C , Kishimoto.T.K and Butcher,E.C. (1991) The peripheral lymph node homing receptor LECAM-1 is involved in CD18-independent adhesion of human neutrophils to endothelium. Biochem. Biophys. Res. Commun., 174, 236-243. Handa,K., Nudelman.E.D., Stroud,M.R., Shiozawa.T and Hakomon.S (1991) Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Le" and sialosyl-Le", and sulfated glycans modulate this binding. Biochem. Biophvs. Res. Commun., 181, 1223-1230. Imai.Y.. True.D.D., Singer.M.S. and Rosen,S.D. (1990) Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor. J Cell Biol., I l l , 1225-1232. Johnston.G.I.. Cook.R.G. and McEver.R.P. (1989) Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell, 56, 1033—1044. Larsen.E., Palabrica.T., Sajer,S.. Gilbert,G.E., Wagner,D.D.. Furie.B.C. and Furie.B. (1990) PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III (CD15). Cell, 63, 4 6 7 ^ 7 4 . Lasky,L.S , Singer.M.S., Yednock.T.A.. Dowbenko.D , Fennie.C . Rodriguez,H.. Nguyen,T., Stachel.S. and Rosen,S.D. (1989) Cloning of a lymphocyte homing receptor reveals a lectin domain. Cell, 56. 1045-1055. Lawrence.M.B. and Spnnger.T.A. (1991) Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell, 65, 859-873. Loveless.R.W., Floyd-O'Sullivan,G., Raynes,J.G.. Yuen,C.-T. and Feizi.T (1992) Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose. /V-acetylgalactosamine and glucuronic acid. EMBO J . 11. 813-819. Lowe.J.B.. Stoolman.L.M., Nair,R.P., Larens.R.D.. Berhend.T.L. and Marks.R.M. (1990) ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell. 63. 475-484.
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References
B)
Glyco-Forum section
Biochemical Society Carbohydrate Group Colloquia December 17-18, 1992; Royal Free Hospital, London 'Carbohydrates, Shapes and Biological Recognition' In honour of Professor Bill Whelan (Miami). Speakers will include: Bill Whelan (Miami), Phil Cohen (Dundee), Louise Johnson (Oxford), Mirek Cygler (Montreal), Florante Quiocho (Houston), Steve Homans (Dundee), Chris Edge (Oxford), Kurt Drickamer (New York), Y.C.Lee (Baltimore), Ten Feizi (London), Ken Reid (Oxford), Maureen Taylor (Oxford), John Summerfield (London), John Gallagher (Manchester). Professor Sen-Itorih Hakomori will deliver the Morton Lecture of the Biochemical Society at the Royal Free Meeting on the evening of December 16th. For further information contact: Anne Dell, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ. Tel: (071) 225 8263 Fax: (071) 225 0458 Joint Meeting of the Carbohydrate Groups of the Royal Society of Chemistry and the Biochemical Society 'Structure, Function and Synthesis of Bio-active Oligosaccharides' March 29 to April 1, 1993 at the University of Dundee. Organizers: John Brimacombe, Michael Ferguson and Steven Homans (University of Dundee). GLYCO XII—International Symposium on Glycoconjugates
Meeting Announcements Keystone Symposia on Molecular & Cellular Biology Announces: Cell Adhesion Mechanisms in Leukocyte Traffic Organizer: Michael Gallatin January 24-31, 1993; Keystone, Colorado Sponsored by ICOS Corporation Carbohydrate Ligands and Their Protein Receptors: Biological Function and Molecular Interaction Organizer: Roger A.Laine January 24—31, 1993; Keystone, Colorado Sponsored by Hoffmann-La Roche, Inc. and R.W.Johnson Pharmaceutical Research Institute For complete programs and application information, please contact: Richard D.Handy Promotion Manager Keystone Symposia Drawer 1630 Silverthorne, CO 80498, USA Phone: (303) 262-1230 Fax: (303) 262-1525
August 15-20, 1993 in Krakow, Poland Organizing committee: J. Koscielak — Chairman T.Chojnacki D.Hoja E.Lisowska A.Lityriska W.Ostrowski P.Strzyga M.Ugorski E.Zdebska International scientific committee: J.Koscielak —Chairman (Poland) S.C.Basu (USA) T.Chojnacki (Poland) R.C.Hughes (UK) A.Kobata (Japan) U.P.F.Lindhal (Sweden) E.Lisowska (Poland) R.Schauer (Germany) N.Sharon (Israel) V.N.Shibaev (Russia) A.Verbert (France) J.F.G.Vliegenthart (The Netherlands) 399
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Luscinskas.F.W., Brock,A.F.. Arnaout,M.A. and Gimbrone.M.A.Jr (1989) Endothelial-leukocyte adhesion molecule-1 dependent and leukocyte (CDll/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium. J. Immunol., 142, 2257-2263. Phillips,M.L.. Nudelman.E., Gaeta.F.C.A., Perez.M., Singhal.A.K., Hakomori,S. and Paulson.J.C. (1990) ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le\ Science, 250, 1130-1132. Polley,M.J.. Philhps.M.L., Wayner,E., Nudelman,E., Singhal.A.K., Hakomon,S. and Paulson.J.C. (1991) CD62 and endothehal cell-leukocyte adhesion molecule (ELAM-1) recognize the same carbohydrate ligand, sialylLewis x. Proc. Natl. Acad. Sci. USA, 88, 6224-6228. Roberts,D.D., Rao,C.N., Liotta,L.A., Gralnick.H.R. and Ginsburg.V. (1986) Comparison of the specificities of laminin, thrombospondin and von Willebrand factor for binding to sulfated glycolipids. J. Biol. Chem., 261. 6872-6877. Skjnner,M.P., Fournier.D J., Andrews.R.K., Gorman.J.J.. Chesterman.C.N. and Berndt,M.C. (1989) Characterization of human platelet GMP-140 as a heparin-binding protein Biochem. Bwphys. Res. Commun., 164. 1371-1379. Skinner,M.P., Lucas,CM., Burns,G.F.. Chesterman.C.N. and Berndt.M.C. (1991) GMP-140 binding to neutrophils is inhibited by sulfated glycans. J. Biol. Chem., 266, 5371-5374 Stoolman, L.M. and Rosen.S.D. (1983) Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation. J. Cell Biol., 96, 722-729. TedderJ.F., Isaacs,G M., Ernst.T.J.. Demetn,G.D., Adler,D A. and Disteche.C.M. (1989) Isolation and chromosomal localization of cDNA's encoding a novel lymphocyte cell surface molecule, LAM-1. J. Exp. Med., 170, 123-133. Tiemeyer.M., Swiedler.S.J., Ishihara.M., Moreland.M., Schweingruber,H., Hirtzer.P and Brandley,B.K. (1991) Carbohydrate ligands for endothelialleukocyte adhesion molecule 1. Proc. Natl. Acad. Sa. USA, 88, 1138-1142. Tyrrell,D., James,P., Rao.N., Foxall.C, Abbas,S., Dasgupta.R., Nashed,M., Hasegawa.A., Kiso.M , Asa.D., Kidd.J. and Brandley.B.K. (1991) Structural requirements for the carbohydrate ligand of E-Selectin. Proc. Nail. Acad. Sa USA. 88. 10372-10376. Walz,G., Aruffo.A., Kolanus,W , Bevilacqua.M. and Seed.B. (1990) Recognition by ELAM-1 of the sialyl-Le" determinant on myeloid and tumor cells. Science, 250, 1132-1135. Watson,S.R., Fennie.C and Lasky.L.A. (1991) Neutrophil influx into an inflammatory site inhibited by a soluble homing receptor-IgG chimaera. Nature, 349, 164-167
1 5. OCT
Glyco-Forum section Letter to the Glyco-Forum Evidence for two classes of carbohydrate binding sites on selectins Darwin Asa, Tracey Gant, Yuko Oda and Brian K.Brandley Glycomed, Inc., 860 Atlantic Avenue, Alameda. CA 94501, USA
Sialyl Lewis x ligands The ability of the selectins to bind to sialyl Lewis x oligosaccharides has been previously reported by a number of groups, using several different techniques. There is now general agreement that E-Selectin recognizes NeuNAc a2-3 Gal/31-4 (Fuccd-3) GlcNAc (sialyl Lewis x, or sLex) and related oligosaccharides (Lowe et al., 1990; Phillips et al., 1990; Walz et al., 1990; Berg et al., 1991; Tiemeyer et al., 1991; Tyrrell et al., 1991). P-Selectin has been reported to bind to the Lewis x structure (Gal /31-4 (Fucal-3) GlcNAc) (Larsen et al., 1990) and/or sLe" (Polley et al., 1991; Foxall et al., 1992). Recently we reported that L-Selectin can also recognize the sLe* structure (Foxall et al., 1992; Figure 1A). Selectin binding to sLe" is dependent on divalent cations, but not exclusively calcium (Figure 2). Strontium is capable of replacing calcium to restore binding of E-Selectin to sLex. For L-Selectin, copper, in addition to strontium, can substitute for calcium. When using P-Selectin, strontium and magnesium (partially) can restore binding to sLe". This effect of magnesium on P-Selectin binding to synthetic sLex is consistent with the observations of Geng et al. (1990) who reported that magnesium could stimulate P-Selectin-mediated neutrophil adhesion at suboptimal levels of calcium. Oxford University Press
Carbohydrates as dissimilar as yeast phosphomannan (PPME), an algal polysaccharide containing fucose sulphate (fucoidan) and sulphatides were used as tools to examine L-Selectin binding activity (Stoolman and Rosen, 1983; Imai et al., 1990), even before the selectins were recognized as a class of related proteins. In addition to sialyl Lewis x, all three selectins bind to sulphatides [Figure IB and Imai et al. (1990)], and L- and P-Selectins bind to SulphoGlucuronylNeoLacto (SGNL) lipid, an HNK-1 reactive epitope (Figure 1C). The direct binding of L- and P-Selectins to SGNL lipid is the clearest demonstration of the similarity of these two receptors, as well as their functional separation from E-Selectin. All the carbohydrate structures recognized by the selectins possess negative charge, but despite this they are very structurally dissimilar. The specificity of these interactions is indicated by three lines of evidence, (i) The oligosaccharide charge alone is not sufficient for recognition since many negatively charged structures (both carbohydrate and non-carbohydrate in nature) are not recognized, (ii) Antibodies known to block selectin function can inhibit the binding of the appropriate selectin to each of the recognized carbohydrate structures (unpublished observations), (iii) Proteins such as human IgG and CD4—IgG chimera do not bind to these carbohydrates, controlling for any artifactual binding of the chimera constructs. The presence of a sulphate ester and uronic acid suggests a similarity between SGNL lipid and glycosaminoglycans (GAGs). GAGs containing primarily iduronic acids (heparin and dermatan sulphate) inhibit L- and P-Selectin binding to sLex and sulphatides (Table I). Other GAGs (chondroitin sulphate, keratan sulphate and Escherichia coli K5) are less potent inhibitors or are ineffective at inhibiting selectin binding. Selectin binding to SGNL appears to be less sensitive to inhibition by GAGs (unpublished observations), but heparin and dermatan sulphate were still more effective than chondroitin sulphate, keratan sulphate or K5. In addition, L- and P-Selectin bind to heparin adsorbed to microtitre plates, or immobilized on agarose beads, in a calcium-independent manner (unpublished observations). E-Selectin is not inhibited by any GAG tested and does not bind to heparin affinity columns (unpublished observations). There are conflicting reports in the literature as to the ability of GAGs to inhibit selectin binding. Rosen's group reported the ability of heparin to inhibit L-Selectin activity in vitro at 20-100 /tg/ml (Stoolman and Rosen, 1983; Imai et al., 1990), but heparin was inactive in other studies when used at 25 jtg/ml. Several laboratories have used heparin at from 100 /tg/ml to 5 mg/ml to block P-Selectin activity, but data from only a single heparin concentration are reported (Aruffo et al., 1991; Handa et al., 1991; Skinner et al., 1991). Skinner et al. (1989) describe the ability of P-Selectin to bind to a heparin affinity column. They also report that the interaction of heparin with P-Selectin is calcium independent (see above). Our data indicate that at least under certain assay conditions, heparin can block L- and P-Selectin function at relatively low concentrations.
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The selectins (LEC-CAMs) are a family of three homologous receptors implicated in the initial interactions between leukocytes and vascular endothelia leading to lymphocyte homing, platelet binding and neutrophil extravasation (Luscinskas et al., 1989; Hallman et al., 1991; Lawrence and Springer, 1991; Watson et al., 1991). E-Selectin (LECAM-2, ELAM-1), L-Selectin (LECAM-1, LAM-1, gp90MEL) and P-Selectin (LECAM-3, GMT-140, PADGEM) each contain a domain with homology to calcium-dependent lectins (Bevilacqua et al., 1989; Johnston et al., 1989; Lasky et al., 1989; Tedder et al., 1989), which has led to an intense effort to define carbohydrate ligands for these proteins. Sialyl Lewis x. (NeuNAc a2-3 Gal/31-4 (Fuc a 1-3) GlcNAc) has been identified as a potential ligand for all three selectins (Figure 1A), but this simple onereceptonone-ligand interaction may not explain the complex biology of the receptors. While differential expression of receptors or ligands may account for the observed biological specificity, we feel that there is significant evidence suggesting that multiple carbohydrate binding sites exist on the selectins.
Carbohydrate ligands unrelated to sialyl Lewis x
Glyco-Forum section
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Fig. 1. Selectin adhesion to glycolipids. The adhesion of selectms to adsorbed glycolipid was performed by ELISA as described in Foxall el al. (1992). Briefly, 25 pmol/well glycolipid were adsorbed to microtitre wells overnight. The plates were rinsed and then blocked with 5% bovine serum albumin solution for 1 h. After blocking, plates were rinsed and selectin—IgG chimera, biotinylated anti-human IgG and Streptavidin-AP were added to glycolipid-coated plates and incubated for 45 mm at 37 °C. After binding, the plate was rinsed and binding read using AP substrate at 405 nm. (A) Adhesion of selectins to 2,3 sLe* glycolipid. (B) Adhesion of selectins to sulphatides. (C) Adhesion of selectins to SGNL lipid. Closed symbols in A and B represent adhesion to 2,6 sLe* glycolipid.
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Fig. 2. Effect of Ca replacement by other divalent cations on selectin binding. Buffers used in the ELISA assay as described in Foxall el al. (1992) were pretreated to remove divalent cations by passing them over a Chelex 100 (Bio-Rad) column. After cation depletion, divalent cations (at concentrations from left to right of 2.5. 1.25 and 0.625 mM) were added back to cation-depleted buffers and the ability of those cations to support selectin binding to 2.3 sLe* glycolipid was assessed using the previously described ELISA procedure. (A) E-Selectin; (B) L-Selectin: (C) P-Selectin.
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Glyco-Forum section
Table I. Inhibition of selectin binding by GAGs. The ability of several GAGs to inhibit the binding of selectins was assayed using the previously described EL1SA. GAGs were incubated with the selectin complex for 45 min prior to the addition of selectin to glycolipid-coated wells. Only data on L- and P-Selectin are presented as GAGs do not inhibit E-Selectm binding L-Selectin Surface (% of control binding) GAG Heparin
[GAG] (Mg/ml) 10 1 01 0.01
2,3 sLe*
Sulphatides
25 2 ± 2.5
37.5 ± 3.7
56.1 ± 3.4 81 9 ± 2 8 88 ± 2.6
70.3 ± 1.2 93.1 ± 1.8 91.8 ± 6.1
92.8 76 6 87 2 90.1
+ 7.8 ± 2.2 ± 7.3 ±9
111 6 105.2 102 5 123 7
± ± ± ±
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100 10 1 0.1
23 7 46.4 103 112.6
± 2.5 ±49 ± 9.8 ± 9.1
45.3 87.2 110.3 115.1
±39 ±38 ±47 ± 3.1
K5 (E.coh)
100 10 1 0.1
101.3 101 106 105.6
±42 ± 2 ± 1.2 ±-113
1147 102.1 102 8 121 1
± ± ± ±
12.7 4.8 52 9.2
Keratan sulphate
250 125 62.5 31.25
115.7 127 5 104.3 106.3
± ± ± ±
94.2 107.2 112.6 98.9
± ± ± ±
9.1 10.5 4.1 3.5
3.1 5.3 7.3 5.1
15.6 5.8 0.8 5.5
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[GAG] (/ig/ml)
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± + ± ±
1.1 1.5 2.3 5.9
20.6 19.1 42 3 89.7
± ± ± ±
0.7 0.8 3.4 4.2
250 125 62.5 31.25
14.1 25.9 33.9 55.5
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32.9 40 67.9 73.9
± ± ± ±
1.3 1 8.3 1.4
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8.7 9.6 17.6 92.6
± ± + ±
0.7 0.4 0.2 5.9
12.7 15.3 28.6 79
± + ± ±
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± ± ± ±
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± ± ± ±
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Chond sulphate
The binding of all selectins to sLe\ and E-Selectin binding to sulphatides, is calcium dependent. However, the binding of L- and P-Selectins to sulphatides and SGNL lipid is only partially blocked by the removal of calcium and the addition of as little as 100 nM EDTA. The addition of increasing amounts of EDTA shows little further reduction up to 5 mM EDTA (Figure 3). This biphasic response to EDTA suggests two modes of interaction of these selectins with sulphatides and SGNL lipid: one calcium dependent and one calcium independent. L-Selectin binding to fucoidan, and L- and P-Selectin binding to heparin, are also largely calcium-independent recognition events (unpublished data).
Specificity Since the selectins seem to recognize several oligosaccharides by what may be multiple binding sites, one may reasonably question how to define specific carbohydrate binding. By the narrowest definition, specific recognition may require calciumdependent binding of structurally related oligosaccharides to one site in the C-lectin domain. However, many mammalian lectins are known that do not require divalent cations for carbohydrate binding (Drickamer, 1988). Furthermore, many proteins not normally considered lectins do bind carbohydrates that include heparin and sulphatides. Several proteins without identified lectin domains have been reported to bind to sulphatides (Roberts et ai, 1986), including laminin, von Willebrand factor (vWF) and thrombospondin. Others have been reported to bind to SGNL epitope (Loveless et al., 1992) and heparin (Burgess and Maciag, 1989). Such calciumindependent carbohydrate recognition has been considered specific because relatively well-defined structural elements of the carbohydrates were required. By this broader definition, sulphatide and SGNL lipid binding to selectins may be considered specific, even though it is calcium independent. So there is precedent to suggest that portions of the selectins, perhaps outside of the C-lectin domain (i.e. EGF or CBP-like domains), may mediate binding to carbohydrates distinct from those recognized by the lectin domain.
Are there two carbohydrate binding sites on L- and P-Selectin? All data currently available indicate that E-Selectin recognizes sLe" and related carbohydrates by a mechanism that is completely calcium dependent. The story for L- and P-Selectins seems more complex. The ability of these selectins to bind to varied carbohydrate structures suggests that more than one binding site may be involved. Several experiments presented here support this view, (i) The binding of L- and P-Selectins to sulphatides, SGNL lipid and heparin exhibits a biphasic response to the addition of EDTA. This is in contrast to selectin binding to sLe", which is completely blocked with very low levels of EDTA. (ii) The pH optimum for binding to sLex is 7, while the optimum for binding to sulphatides is 5 (unpublished observation), again suggesting different sites, (iii) Binding of selectins to sulphatides is relatively insensitive to increased 397
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250 125 62.5 31.25
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Cation chelation
Glyco-Forum section
that L- and P-Selectins bind to sulphatides and SGNL lipid by a site that is functionally distinct from the sLex binding site. It is unclear whether both sites reside in the lectin domain, or if the calcium-independent binding site resides in the EGF or CBP-like domains. Inhibition of binding to all putative ligands by antibodies and polysaccharides suggests that the two sites interact in some way, either being physically contiguous or overlapping, or by induction of a conformational change at distant sites (Handa et ai, 1991). We feel that the possibility of mutiple sugar binding sites on selectins may help explain their biological activity. Identification of a second carbohydrate binding site on selectins provides another target for the design of carbohydrate-based inhibitors of the selectins.
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ionic strength (up to 500 mM) compared to binding to sLe\ which exhibits optimal binding at 150—200 mM NaCl (unpublished observation), (iv) Cation inhibitors also discriminate between sLex and sulphatide binding. Barium (at 1 mM) can inhibit the binding of all three selectins to sLe\ but it is relatively ineffective at blocking selectin binding to sulphatides (unpublished observations). Taken together, these data suggest 398
Aruffo.A., Kolanus,W., Walz.G.. Fredman,P. and Seed.B. (1991) CD62/ P-Selectin recognition of myeloid and tumor cell sulfatides Cell, 67, 35—44. Berg.E.L . Robinson,M.K., Mansson.O., Butcher,E.C. and Magnani.J.L (1991) A carbohydrate domain common to both sialyl Le" and sialyl Le* is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1 J. Biol. Chem.. 265, 14869-14872. Bevilacqua,M.P., Stengelin,S., Gimbrone,M.A.,Jr and Seed.B. (1989) Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins Science, 243. 1160-1165. Burgess,W.H and Maciag.T (1989) The Heparin-Binding Growth Factor family of proteins. Annu. Rev. Biochem.. 58, 575—606. Drickamer,K. (1988) Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem., 263, 9557-9560. Foxall.C.F., Watson,S.R., Dowbenko.D., Fenme.C, Lasky.L.A., Kiso,M.. Hasegawa.A., Asa,D. and Brandley.B.K. (1992) The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis x oligosaccharide. J. Cell Biol., in press. Geng,J.-G., Moore,K.L., Johnson.A.E. and McEver,R.P. (1991) Neutrophil recognition requires a Ca2+-induced conformational change in the lectin domain of GMP-140. J. Biol. Chem., 266. 22313-22318. Hallman,R , Jutila,M.A., Smith,C.W , Anderson,D C , Kishimoto.T.K and Butcher,E.C. (1991) The peripheral lymph node homing receptor LECAM-1 is involved in CD18-independent adhesion of human neutrophils to endothelium. Biochem. Biophys. Res. Commun., 174, 236-243. Handa,K., Nudelman.E.D., Stroud,M.R., Shiozawa.T and Hakomon.S (1991) Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Le" and sialosyl-Le", and sulfated glycans modulate this binding. Biochem. Biophvs. Res. Commun., 181, 1223-1230. Imai.Y.. True.D.D., Singer.M.S. and Rosen,S.D. (1990) Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor. J Cell Biol., I l l , 1225-1232. Johnston.G.I.. Cook.R.G. and McEver.R.P. (1989) Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell, 56, 1033—1044. Larsen.E., Palabrica.T., Sajer,S.. Gilbert,G.E., Wagner,D.D.. Furie.B.C. and Furie.B. (1990) PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III (CD15). Cell, 63, 4 6 7 ^ 7 4 . Lasky,L.S , Singer.M.S., Yednock.T.A.. Dowbenko.D , Fennie.C . Rodriguez,H.. Nguyen,T., Stachel.S. and Rosen,S.D. (1989) Cloning of a lymphocyte homing receptor reveals a lectin domain. Cell, 56. 1045-1055. Lawrence.M.B. and Spnnger.T.A. (1991) Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell, 65, 859-873. Loveless.R.W., Floyd-O'Sullivan,G., Raynes,J.G.. Yuen,C.-T. and Feizi.T (1992) Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose. /V-acetylgalactosamine and glucuronic acid. EMBO J . 11. 813-819. Lowe.J.B.. Stoolman.L.M., Nair,R.P., Larens.R.D.. Berhend.T.L. and Marks.R.M. (1990) ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell. 63. 475-484.
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References
B)
Glyco-Forum section
Biochemical Society Carbohydrate Group Colloquia December 17-18, 1992; Royal Free Hospital, London 'Carbohydrates, Shapes and Biological Recognition' In honour of Professor Bill Whelan (Miami). Speakers will include: Bill Whelan (Miami), Phil Cohen (Dundee), Louise Johnson (Oxford), Mirek Cygler (Montreal), Florante Quiocho (Houston), Steve Homans (Dundee), Chris Edge (Oxford), Kurt Drickamer (New York), Y.C.Lee (Baltimore), Ten Feizi (London), Ken Reid (Oxford), Maureen Taylor (Oxford), John Summerfield (London), John Gallagher (Manchester). Professor Sen-Itorih Hakomori will deliver the Morton Lecture of the Biochemical Society at the Royal Free Meeting on the evening of December 16th. For further information contact: Anne Dell, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ. Tel: (071) 225 8263 Fax: (071) 225 0458 Joint Meeting of the Carbohydrate Groups of the Royal Society of Chemistry and the Biochemical Society 'Structure, Function and Synthesis of Bio-active Oligosaccharides' March 29 to April 1, 1993 at the University of Dundee. Organizers: John Brimacombe, Michael Ferguson and Steven Homans (University of Dundee). GLYCO XII—International Symposium on Glycoconjugates
Meeting Announcements Keystone Symposia on Molecular & Cellular Biology Announces: Cell Adhesion Mechanisms in Leukocyte Traffic Organizer: Michael Gallatin January 24-31, 1993; Keystone, Colorado Sponsored by ICOS Corporation Carbohydrate Ligands and Their Protein Receptors: Biological Function and Molecular Interaction Organizer: Roger A.Laine January 24—31, 1993; Keystone, Colorado Sponsored by Hoffmann-La Roche, Inc. and R.W.Johnson Pharmaceutical Research Institute For complete programs and application information, please contact: Richard D.Handy Promotion Manager Keystone Symposia Drawer 1630 Silverthorne, CO 80498, USA Phone: (303) 262-1230 Fax: (303) 262-1525
August 15-20, 1993 in Krakow, Poland Organizing committee: J. Koscielak — Chairman T.Chojnacki D.Hoja E.Lisowska A.Lityriska W.Ostrowski P.Strzyga M.Ugorski E.Zdebska International scientific committee: J.Koscielak —Chairman (Poland) S.C.Basu (USA) T.Chojnacki (Poland) R.C.Hughes (UK) A.Kobata (Japan) U.P.F.Lindhal (Sweden) E.Lisowska (Poland) R.Schauer (Germany) N.Sharon (Israel) V.N.Shibaev (Russia) A.Verbert (France) J.F.G.Vliegenthart (The Netherlands) 399
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Luscinskas.F.W., Brock,A.F.. Arnaout,M.A. and Gimbrone.M.A.Jr (1989) Endothelial-leukocyte adhesion molecule-1 dependent and leukocyte (CDll/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium. J. Immunol., 142, 2257-2263. Phillips,M.L.. Nudelman.E., Gaeta.F.C.A., Perez.M., Singhal.A.K., Hakomori,S. and Paulson.J.C. (1990) ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le\ Science, 250, 1130-1132. Polley,M.J.. Philhps.M.L., Wayner,E., Nudelman,E., Singhal.A.K., Hakomon,S. and Paulson.J.C. (1991) CD62 and endothehal cell-leukocyte adhesion molecule (ELAM-1) recognize the same carbohydrate ligand, sialylLewis x. Proc. Natl. Acad. Sci. USA, 88, 6224-6228. Roberts,D.D., Rao,C.N., Liotta,L.A., Gralnick.H.R. and Ginsburg.V. (1986) Comparison of the specificities of laminin, thrombospondin and von Willebrand factor for binding to sulfated glycolipids. J. Biol. Chem., 261. 6872-6877. Skjnner,M.P., Fournier.D J., Andrews.R.K., Gorman.J.J.. Chesterman.C.N. and Berndt,M.C. (1989) Characterization of human platelet GMP-140 as a heparin-binding protein Biochem. Bwphys. Res. Commun., 164. 1371-1379. Skinner,M.P., Lucas,CM., Burns,G.F.. Chesterman.C.N. and Berndt.M.C. (1991) GMP-140 binding to neutrophils is inhibited by sulfated glycans. J. Biol. Chem., 266, 5371-5374 Stoolman, L.M. and Rosen.S.D. (1983) Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation. J. Cell Biol., 96, 722-729. TedderJ.F., Isaacs,G M., Ernst.T.J.. Demetn,G.D., Adler,D A. and Disteche.C.M. (1989) Isolation and chromosomal localization of cDNA's encoding a novel lymphocyte cell surface molecule, LAM-1. J. Exp. Med., 170, 123-133. Tiemeyer.M., Swiedler.S.J., Ishihara.M., Moreland.M., Schweingruber,H., Hirtzer.P and Brandley,B.K. (1991) Carbohydrate ligands for endothelialleukocyte adhesion molecule 1. Proc. Natl. Acad. Sa. USA, 88, 1138-1142. Tyrrell,D., James,P., Rao.N., Foxall.C, Abbas,S., Dasgupta.R., Nashed,M., Hasegawa.A., Kiso.M , Asa.D., Kidd.J. and Brandley.B.K. (1991) Structural requirements for the carbohydrate ligand of E-Selectin. Proc. Nail. Acad. Sa USA. 88. 10372-10376. Walz,G., Aruffo.A., Kolanus,W , Bevilacqua.M. and Seed.B. (1990) Recognition by ELAM-1 of the sialyl-Le" determinant on myeloid and tumor cells. Science, 250, 1132-1135. Watson,S.R., Fennie.C and Lasky.L.A. (1991) Neutrophil influx into an inflammatory site inhibited by a soluble homing receptor-IgG chimaera. Nature, 349, 164-167
