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L-selectin Interactions with Novel Mono- and Multisulfated Lewisx Sequences in Comparison with the Potent Ligand 3′-Sulfated Lewisa

1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês

10.1074/jbc.274.26.18213

1083-351X

Christine Galustian, André Lubineau, Christine Le Narvor, Makoto Kiso, Gavin M. Brown, Ten Feizi,

Immune Response and Inflammation

The cell adhesion molecule L-selectin binds to 3′-sialyl-Lewis (Le)x and -Lea and to 3′-sulfo-Lex and -Lea sequences. The binding to 3′-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on theN-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6′-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3′-sulfo-Lex, the 6-sulfation enhances and 6′-sulfation suppresses L-selectin binding. The 6′-sulfation without 3′-sialyl or 3′-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3′-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and innerN-acetylglucosamine do not influence the binding. Although binding to the 6,3′-sulfo-Lex and 6-sulfo,3′-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3′-sulfo-Lex series are more potent than the corresponding 3′-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3′-sulfo-Lex (and also the 3′-sulfo-Lea) would seem most appropriate. The cell adhesion molecule L-selectin binds to 3′-sialyl-Lewis (Le)x and -Lea and to 3′-sulfo-Lex and -Lea sequences. The binding to 3′-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on theN-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6′-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3′-sulfo-Lex, the 6-sulfation enhances and 6′-sulfation suppresses L-selectin binding. The 6′-sulfation without 3′-sialyl or 3′-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3′-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and innerN-acetylglucosamine do not influence the binding. Although binding to the 6,3′-sulfo-Lex and 6-sulfo,3′-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3′-sulfo-Lex series are more potent than the corresponding 3′-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3′-sulfo-Lex (and also the 3′-sulfo-Lea) would seem most appropriate. L-selectin, a carbohydrate-binding adhesion molecule on leukocytes that binds to saccharide ligands on high endothelial cells in post-capillary venules of lymph nodes, has a key role in the initial stages of leukocyte extravasation into peripheral lymph nodes and areas of acute and chronic inflammation (1Rosen S.D. Histochemistry. 1993; 100: 185-191Crossref PubMed Scopus (48) Google Scholar, 2Tedder T.F. Steeber D.A. Pizcueta P. J. Exp. Med. 1995; 181: 2259-2264Crossref PubMed Scopus (396) Google Scholar). Previous work with structurally defined oligosaccharides has shown that L-selectin binds to Lewisa(Lea) 1The abbreviations used are: Lea and Lex, Lewisa and Lewisx.and Lex sequences sialylated or sulfated at position 3 of outer galactose with a preference for binding to 3′-sialyl-Leaover 3′-sialyl-Lex (3Berg E.L. Magnani J. Warnock R.A. Robinson M.K. Butcher E.C. Biochem. Biophys. Res. Commun. 1992; 184: 1048-1055Crossref PubMed Scopus (173) Google Scholar) and a preference of 3′-sulfo-Lea and 3′-sulfo-Lex over the sialyl forms (4Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (175) Google Scholar, 5Green P.J. Yuen C.-T. Childs R.A. Chai W. Miyasaka M. Lemoine R. Lubineau A. Smith B. Ueno H. Nicolaou K.C. Feizi T. Glycobiology. 1995; 5: 29-38Crossref PubMed Scopus (79) Google Scholar, 6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar). The occurrence of 3′-sulfated forms of Lexand Lea has been documented on epithelial glycoproteins, and this led to the demonstration that sulfate can substitute effectively for sialic acid in ligands for the E- and L-selectins (4Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (175) Google Scholar,7Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (239) Google Scholar, 8Chai W. Feizi T. Yuen C.-T. Lawson A.M. Glycobiology. 1997; 7: 861-872Crossref PubMed Scopus (82) Google Scholar). Among these four sequences, the strongest binding signal is with the 3′-sulfo-Lea (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar). These findings are important for the design of synthetic, potentially therapeutic analogs of the selectin ligands, as chemical synthesis of sulfated oligosaccharides is far more facile than of sialyl-oligosaccharides. O-glycosidic oligosaccharides with other sulfation patterns have been isolated from one of the counter-receptors of L-selectin, GlyCAM-1; these are heptasaccharides with 3′-sialyl-Lex capping groups containing 6-O-sulfate at the outer galactose (referred to as 6′-O-sulfation), at the penultimateN-acetylglucosamine (6-O-sulfation), or at both of these positions (6′,6-O-sulfation) (9Hemmerich S. Rosen S.D. Biochemistry. 1994; 33: 4830-4835Crossref PubMed Scopus (184) Google Scholar). We have demonstrated that the 6-sulfo,3′-sialyl-Lex sequence constitutes a strong ligand for L-selectin, particularly where theN-acetylneuraminic acid is de-N-acetylated, whereas the 6′-sulfated analog is not bound (10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar, 24Komba S. Galustian G. Ishida H. Feizi T. Kannagi R. Kiso M. Angew. Chem. Int. Ed. Engl. 1999; 38: 1131-1133Crossref PubMed Scopus (77) Google Scholar). Indeed, the addition of 6′-O-sulfate to the 6-sulfo,3′-sialyl-Lex sequence impairs the L-selectin binding. Knowing that 3-O-sulfation at galactose of Lexor Lea can substitute for 3′-sialylation in the formation of saccharide motifs recognized by the selectins, we have explored in the present study the reactivities of human L-selectin with a novel series of mono- and multisulfated Lex sequences in which the 3′-sialyl residue on Lex is replaced by 3′-O-sulfate, and we make a comparison with reactivity toward the 3′-sulfo-Lea sequence, which is among the most potent L-selectin ligands thus far described, and also with reactivity toward the 6′-sulfo-Lex. We report here results that reveal an advantage of 3′-sulfation over 3′-sialylation of Lexwith respect to inhibitory activity toward L-selectin binding and the deleterious effect of 6′-sulfation of Lex with respect both to binding and inhibitory activity toward L-selectin binding. We also examine L-selectin binding to a novel trisulfated sequence, 3′-sialyl,6-sulfo-di-N-acetyl lactosamine, with two additional sulfates, one at the inner galactose and another at the inner N-acetylglucosamine residue (both at position 6). We show that the additional sulfates along the extended backbone do not influence the binding signal. The carbohydrate sequences investigated are shown in TableI. The following were synthesized chemically: the 3′-sulfo-Lea (11Lubineau A. Le-Gallic J. Lemoine R. Bioorg. Med. Chem. Lett. 1994; 2: 1143-1151Crossref Scopus (27) Google Scholar); the 3′-sulfo-Lex, the 6′,3′-sulfo-Lex; the 6,3′-sulfo-Lex; the 6′-sulfo-Lex (12Auge C. Dagron F. Lemoine R. Le Narvor C. Lubineau A. Chapleur Y. Carbohydrate Mimics: Concepts and Methods. Verlag Chemie, Weinhein, Germany1997: 365-383Google Scholar); and also the 3′-sialyl-Lexpentasaccharide, 2For ease of comparison of the sialyl-Lex and -Lea with the sulfo-Lex and -Lea oligosaccharides, the term pentasaccharide is used to denote the fucosyl tetrasaccharide backbones that they share. GSC151, (13Hasegawa A. Ando T. Kameyama A. Kiso M. J. Carbohydr. Chem. 1992; 11: 645-658Crossref Scopus (41) Google Scholar). These were purified by high pressure liquid chromatography using a TSK-Gel® amide 80 column (Tosohaas) (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar). Lacto-N-tetraose and lacto-N-neo-tetraose were purchased from Dextra. The oligosaccharide designated C4U was prepared by keratanase II digestion of bovine articular keratan sulfate essentially as described by Brown et al. (14Brown G.M. Huckerby T.N. Morris H.G. Abram B.L. Nieduszynski I.A. Biochemistry. 1994; 33: 4836-4846Crossref PubMed Scopus (54) Google Scholar). The oligosaccharide designated Fuc-C4U was derived from C4U by fucosylation using milk α-(1-3/4)-fucosyltransferase; oligosaccharide identity was corroborated by 1H NMR spectroscopy, and purity was established using high pH anion exchange chromatography. 3G. M. Brown, manuscript in preparation.Neoglycolipids (15Tang P.W. Gooi H.C. Hardy M. Lee Y.C. Feizi T. Biochem. Biophys. Res. Commun. 1985; 132: 474-480Crossref PubMed Scopus (106) Google Scholar) were prepared by conjugation of the above oligosaccharides to the amino phospholipidl-1,2-dihexadecyl-sn-glycerol-3-phosphoethanolamine and purified, and their composition and sequences were corroborated by liquid secondary ion mass spectroscopy as described previously (16Feizi T. Stoll M.S. Yuen C.-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (118) Google Scholar). The following were chemically synthesized glycosylceramides: the 3′-sialyl-Lex (13Hasegawa A. Ando T. Kameyama A. Kiso M. J. Carbohydr. Chem. 1992; 11: 645-658Crossref Scopus (41) Google Scholar); the 6′-sulfo,3′-sialyl-Lex; the 6-sulfo,3′-sialyl-Lex; and the 6′,6-sulfo,3′-sialyl-Lex (17Komba S. Ishida H. Kiso M. Hasegawa A. Bioorg. Med. Chem. 1996; 4: 1833-1847Crossref PubMed Scopus (87) Google Scholar) designated GSC64, GSC268, GSC269, and GSC270, respectively. These were purified by preparative TLC as described previously (10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar).Table ICarbohydrate sequences investigatedDesignationAbbreviationSequence3′-sulfo-Lea3′Su-LeaGalβ1–3GlcNAcβ1–3Galβ1–3Glc‖3 ‖1,4HSO3 Fucα3′-sulfo-Lex3′Su-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖3 ‖1,3HSO3 FucαHSO3‖66′-sulfo-Lex6′Su-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖1,3FucαHSO3‖66′,3′-sulfo-Lex6′,3′-Su-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖3 ‖1,3HSO3 FucαHSO3‖66,3′-sulfo-Lex6,3′-Su-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖3 ‖1,3HSO3 Fucα3′-sialyl-Lex3′S-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖2,3 ‖1,3NeuAcα FucαHSO3‖66′-sulfo,3′-sialyl-Lex6′Su,3′S-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖2,3 ‖1,3NeuAcα FucαHSO3‖66-sulfo,3′-sialyl-Lex6Su,3′S-LexGalβ1–4GlcNAcβ1–3Galβ1–4Glc‖2,3 ‖1,3NeuAcα FucαHSO3 HSO3‖6 ‖66′,6-sulfo,3′-sialyl-Lex6′,6-Su,3′S-LexGalβ1–4GlcNAcβ1–3Galβ1–4-Glc‖2,3 ‖1,3NeuAcα FucαHSO3 HSO3 HSO3‖6 ‖6 ‖6C4UC4UGalβ1–4GlcNAcβ1–3Galβ1–4GlcNAc‖2,3NeuAcαHSO3 HSO3 HSO3‖6 ‖6 ‖6Fuc-C4UFuc-C4UGalβ1–4GlcNAcβ1–3Galβ1–4GlcNAc‖2,3 ‖1,3NeuAcα FucαLacto-N-tetraoseLNTGalβ1–3GlcNAcβ1–3Galβ1–3GlcLacto-N-neo-tetraoseLNNTGalβ1–4GlcNAcβ1–3Galβ1–4Glc Open table in a new tab A soluble form of human-L-selectin fused to the human immunoglobulin (IgG) Fc domain and expressed in transfected Chinese hamster ovary cells (provided by G. Shaw, Genetics Institute Inc., Cambridge, Massachusetts) was isolated from tissue culture supernatant as described previously (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar). This preparation consisted predominantly of tetramers of L-selectin as assessed by gel filtration analysis (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar) and is referred to here as paucivalent. For binding experiments, where indicated, preparations of the multivalent L-selectin were made by incubating for 1 h with goat anti-human immunoglobulin heavy and light chains (Vector) at a selectin:antibody ratio of 1:3 by weight. This ratio was selected from a range, 1:0.5 to 1:10, as it gave the highest binding signal with immobilized reference compounds 3′-sulfo-Lea and 3′-sialyl-Lea. For direct binding experiments (10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar), purified lipid-linked oligosaccharides (glycolipids and neoglycolipids) were immobilized on microwells (Falcon 3912). About 30% of the lipid-linked oligosaccharides added were retained in the microwells under the binding assay conditions (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar). Fifty ng of multivalent L-selectin IgG chimera was applied per well using as diluent 10 mm Tris buffer, pH 7.4, containing 150 mm NaCl and 2 mm Ca2+. As in previous experiments with E-selectin (7Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (239) Google Scholar), binding intensity of L-selectin was of the same order to the 3′-sialyl-Lexsequence in the form of a glycosylceramide or a neoglycolipid (not shown). For inhibition experiments, the immobilized oligosaccharides used were either the lipid-linked 6-sulfo,3′-sialyl-Lex or 6,3′-sulfo-Lex (100 pmol added per well) and 10 ng of the multivalent L-selectin or 50 ng of the paucivalent L-selectin were added per well. These levels of L-selectin were used in the inhibition experiments as they gave comparable binding signals with the two immobilized ligands. Serial dilutions of liposomes containing lipid-linked oligosaccharides were used as inhibitors. These consisted of cholesterol:lecithin:lipid-linked oligosaccharides at ratios of 0.4:0.4:1 by weight. In accord with earlier observations (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar), the 3′-sulfo-Lex sequence was bound by human L-selectin but less strongly than 3′-sulfo-Lea. However, the 6′-sulfo-Lex was not bound (Fig.1 A). The 6-O-sulfation of 3′-sulfo-Lex to give 6,3′-sulfo-Lex elicited enhanced L-selectin binding, whereas the 6′-sulfated analog (6′,3′-sulfo-Lex) consistently showed some diminution of binding. The binding signal observed with the 6,3′-sulfo-Lex sequence was of the same order as that observed with the 6-sulfo,3′-sialyl-Lex (Fig.1 C). Thus, 6-sulfation, but not 6′-sulfation, has a potentiating effect on human L-selectin binding not only to the 3′-sialyl-Lex, as shown previously (Ref. 10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar and Fig.1 B), but also to the 3′-sulfo-Lex sequence. The hindering effect of the 6′-O-sulfation was less pronounced on 3′-sulfo-Lex than on the 3′-sialyl-Lexanalog investigated previously (Fig. 1 B) (10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar). Binding experiments with the oligosaccharide C4U and its 3′-fucosylated analog, Fuc-C4U, clearly showed that the presence of the fucose residue is essential for L-selectin binding to the 3′-sialyl,6-sulfated di-N-acetyllactosamine backbone (Fig. 1 D). The binding signals with the Fuc-C4U and the 6-sulfo,3′-sialyl-Lex were similar, indicating that the additional sulfates on the internal galactose andN-acetylglucosamine residues do not influence the L-selectin binding signal. Inhibition experiments were performed with the multivalent L-selectin using as immobilized ligands the 6-sulfo,3′-sialyl-Lex or the 6,3′-sulfo-Lex(Figs. 2 and3, A and B); all of the acidic compounds of the Lex series tested gave some inhibition of binding. The nonsulfated lacto-N-tetraose gave no inhibition. The 6,3′-sulfo-Lex (IC501.4 × 10−7 and 1.5 × 10−7m with the two immobilized ligands) was approximately 3 and 5 times more active as an inhibitor than the 6-sulfo,3′-sialyl-Lex (5.1 × 10−7 and 6.9 × 10−7m). The least active inhibitors were the nonsulfated 3′-sialyl-Lex(IC50 3.7 × 10−6 and 6.8 × 10−6m), the 6′-sulfo-Lex(3.1 × 10−6 and 2.0 × 10−6m) and the 6′-sulfo,3′-sialyl-Lex (7.4 × 10−6 and 1.0 × 10−6m). Fine comparisons of the inhibitory activities of the relatively potent sequences were difficult as inhibition curves were often closely spaced or partially overlapping as in Fig. 2 B.Figure 3Activities of lipid-linked oligosaccharides of the 3′-sulfo-Lex and 3′-sialyl-Lex series as inhibitors of the binding of human L-selectin to immobilized 6-sulfo, 3′-sialyl-Lex (A and C) and 6,3′sulfo-Lex (B andD). Results of inhibition experiments with the multivalent selectin are shown in A and B and with the paucivalent selectin in C and D. The inhibitory activities are expressed as IC50 values and have been assembled from experiments of which selected inhibition curves only are shown in Figs. 2 and 4. Where results from two experiments are assembled as in A (Fig. 2, taken from experiments 1 and 3) and in C (Fig. 4, taken from experiments 4 and 6), the IC50 values were normalized relative to that of 6,3′-sulfo-Lex, which was included as a reference compound. See Table I for abbreviations.View Large Image Figure ViewerDownload (PPT) Inhibition of binding experiments using the paucivalent L-selectin was performed (Figs. 3, C andD, and 4) to discriminate more clearly (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (43) Google Scholar) between relatively high and low affinity inhibitors and under conditions that may possibly simulate situations in vivo where L-selectin is relatively sparsely expressed. The degree of inhibition of selectin binding depended on the immobilized ligand used; binding to the 6,3′sulfo-Lex was harder to inhibit than to the 6-sulfo,3′-sialyl-Lex (Fig. 3, C andD). Here the 3′-sialyl-Lex series were poorer inhibitors overall than the corresponding 3′-sulfo-Lexanalogs and gave no significant inhibition when the 6,3′-sulfo-Lex was used as the immobilized ligand (Fig.3 D); when the 6-sulfo,3′-sialyl-Lex was used as the immobilized ligand (Fig. 3 C), only the homologous oligosaccharide sequence showed reasonable inhibition (IC501.6 × 10−6m). In contrast, among the oligosaccharides in which there was 3′-sulfate instead of 3′-sialyl, all inhibited the selectin binding to the two immobilized ligands. The 6′-sulfo-Lex was not inhibitory. The 3′-sulfo-Lex and the 6,3′-sulfo-Lex stood out as superior inhibitors with IC50 values of 1.4 × 10−7 and 5.0 × 10−7m, and 5 × 10−8 and 2.7 × 10−7m, respectively. Also included in the present inhibition experiments for comparison was the 3′-sulfo-Lea sequence; its potency was intermediate between that of 3′-sulfo-Lexand 6,3′-sulfo-Lex (Fig. 3, C andD). It is clear from the binding experiments described here, first, that, in contrast to 3′-sulfation of Lex, which supports L-selectin reactivity, the 6′-sulfation does not elicit a detectable binding signal. Second, the L-selectin reactivities of 3′-sulfo- and 3′-sialyl-Lex are similarly influenced by the addition of 6′-sulfate or 6-sulfate. Whereas 6′-sulfation has a negative effect, 6-sulfation has an enhancing effect on L-selectin binding to the clustered, immobilized 3′-sulfo- and 3′-sialyl-Lex series. Thus the 6-sulfate seems to be a part of the recognition motif for L-selectin, whereas 6′-sulfate is not. Moreover, its presence partially hinders the recognition of the 3′-sialyl- or 3′-sulfo-Lexby the selectin. The enhancement of L-selectin binding in the presence of 6-sulfation was substantial for both the 3′-sulfo- and the 3′-sialyl-Lex sequences, but the negative effect of 6′-sulfation was less pronounced with the 3′-sulfo-Lex than with the 3′-sialyl-Lex sequence investigated previously (10Galustian C. Lawson A.M. Komba S. Ishida H. Kiso M. Feizi T. Biochem. Biophys. Res. Commun. 1997; 240: 748-751Crossref PubMed Scopus (78) Google Scholar). Third, whereas the 6-sulfate on the subterminalN-acetylglucosamine of the Lex sequence is clearly a part of the recognition sequence for L-selectin, additional sulfates along the di-N-acetyllactosamine backbone are not recognized. Fourth, the presence of the 3′-linked fucose on 6-sulfo,3′-sialyl-Lex is essential for L-selectin binding. In accord with this finding is an earlier report that L-selectin binding is abolished by modification of the fucose in the 3′-sialyl-Lex sequence by removal of oxygen at position 2, 3, or 5 (18Brandley B.K. Kiso M. Abbas S. Nikrad P. Srivasatava O. Foxall C. Oda Y. Hasegawa A. Glycobiology. 1993; 3: 633-641Crossref PubMed Scopus (208) Google Scholar). The presence of the fucose is apparently less critical in the 3′-sulfo-Lex sequence as it has been observed that the defucosylated 3′-sulfo-Lex tri- and tetrasaccharides are bound by l-selectin, albeit less strongly than the 3′-fucosyl analogs (4Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (175) Google Scholar, 5Green P.J. Yuen C.-T. Childs R.A. Chai W. Miyasaka M. Lemoine R. Lubineau A. Smith B. Ueno H. Nicolaou K.C. Feizi T. Glycobiology. 1995; 5: 29-38Crossref PubMed Scopus (79) Google Scholar). The inhibition experiments described here reveal subtle differences in L-selectin reactivities that are not apparent in the binding experiments, namely a slightly greater binding affinity toward the 3′-sulfo than the 3′-sialyl-Lex. The 6-sulfated 3′-sulfo-Lex sequence had a greater inhibitory activity than the 6-sulfated 3′-sialyl-Lex, although the binding signals they elicited were similar. The differences were more apparent in inhibition experiments where the paucivalent L-selectin was used; these experiments clearly showed differences in the ease of inhibition of L-selectin binding depending on the immobilized ligand used, and they showed that inhibitory activities of the 3′-sulfo-Lexseries were greater than those of the 3′-sialyl-Lex series. Collectively, these data, together with the earlier finding (4Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (175) Google Scholar) that the 3′-sulfo- but not the 3′-sialyl-lactosamine backbone (in the absence of fucose) elicits a binding signal with L-selectin, indicate that the 3′-sulfation at the galactose of Lex creates a higher affinity ligand than the 3′-sialylation and that L-selectin has overall a higher affinity toward the 3′-sulfo- and 6,3′-sulfo-Lex sequences than the corresponding 3′-sialyl-Lex analogs. The inhibition experiments were performed here using as inhibitors oligosaccharides linked to a lipid and displayed on liposomes as it was found earlier that about 10,000-fold less oligosaccharide material is required compared with the free oligosaccharides (6Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. 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When examined in the oligomeric state on dendrimers, IC50 values in the 1–10 μm range were reported (23Roy R. Park W.K.C. Zanini D. Foxall C. Srivastava O.P. Carbohydr. Lett. 1997; 2: 259-266Google Scholar). A general feature that emerges from our inhibition experiments using L-selectin in the multivalent state and those of other groups who have used the multivalent L-selectin is that differences in inhibitory activities of the various acidic Lex and Lea oligosaccharides are less marked. Also, in the multivalent assay, the differing ease of inhibition of L-selectin binding to different immobilized ligands is less readily discernible. The modification to the inhibition assay that we have introduced serves to establish a hierarchy of inhibitory activities that may be relevant in in vivo situations where the display of L-selectin is relatively sparse. Thus, the inhibition assay with paucivalent L-selectin establishes that the 3′-sulfo-Lex series and also the 3′-sulfo-Lea are more effective inhibitors of thel-selectin binding than the 3′-sialyl-Lexanalogs. Among these, the 6,3′-sulfo-Lex and the monosulfated 3′-sulfo-Lea are the most potent inhibitors of L-selectin binding among the oligosaccharide sequences so far tested. The former is only marginally better. Therefore, from the point of view of synthetic strategies for the design of therapeutic oligosaccharide analogs, those based on the relatively simple structure 3-sulfo-Lex and -Lea would seem the most appropriate. We thank Dr. Chun-Ting Yuen and Dr. Colin Herbert for preparation of the neoglycolipids and the purification of the glycosylceramides, Dr. Gray Shaw for thel-selectin-transfected Chinese hamster ovary cell line, Dr. Robert Childs for helpful discussions, and Margaret Runnicles for help in preparation of the manuscript.