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

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 (1,2). Previous work with structurally defined oligosaccharides has shown that L-selectin binds to Lewis a (Le a ) 1 and Le x sequences sialylated or sulfated at position 3 of outer galactose with a preference for binding to 3Ј-sialyl-Le a over 3Ј-sialyl-Le x (3) and a preference of 3Ј-sulfo-Le a and 3Ј-sulfo-Le x over the sialyl forms (4 -6). The occurrence of 3Ј-sulfated forms of Le x and Le a 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 (4,7,8). Among these four sequences, the strongest binding signal is with the 3Ј-sulfo-Le a (6). 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, Gly-CAM-1; these are heptasaccharides with 3Ј-sialyl-Le x capping groups containing 6-O-sulfate at the outer galactose (referred to as 6Ј-O-sulfation), at the penultimate N-acetylglucosamine (6-O-sulfation), or at both of these positions (6Ј,6-O-sulfation) (9). We have demonstrated that the 6-sulfo,3Ј-sialyl-Le x sequence constitutes a strong ligand for L-selectin, particularly where the N-acetylneuraminic acid is de-N-acetylated, whereas the 6Ј-sulfated analog is not bound (10,24). Indeed, the addition of 6Ј-O-sulfate to the 6-sulfo,3Ј-sialyl-Le x sequence impairs the L-selectin binding.
Knowing that 3-O-sulfation at galactose of Le x or Le a can substitute for 3Ј-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 Le x sequences in which the 3Ј-sialyl residue on Le x is replaced by 3Ј-O-sulfate, and we make a comparison with reactivity toward the 3Ј-sulfo-Le a sequence, which is among the most potent L-selectin ligands thus far described, and also with reactivity toward the 6Ј-sulfo-Le x . We report here results that reveal an advantage of 3Ј-sulfation over 3Ј-sialylation of Le x with respect to inhibitory activity toward L-selectin binding and the deleterious effect of 6Ј-sulfation of Le x with respect both to binding and inhibitory activity toward L-selectin binding. We also examine L-selectin binding to a novel trisulfated sequence, 3Ј-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.
Recombinant Soluble L-selectins-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 (6). This preparation consisted predominantly of tetramers of L-selectin as assessed by gel filtration analysis (6) 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 2 For ease of comparison of the sialyl-Le x and -Le a with the sulfo-Le x and -Le a oligosaccharides, the term pentasaccharide is used to denote the fucosyl tetrasaccharide backbones that they share. 3 G. M. Brown, manuscript in preparation.
3Ј-sulfo-Le a and 3Ј-sialyl-Le a . L-selectin Binding and Inhibition Assays-For direct binding experiments (10), 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 (6). 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 Ca 2ϩ . As in previous experiments with E-selectin (7), binding intensity of L-selectin was of the same order to the 3Ј-sialyl-Le x sequence 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,3Ј-sialyl-Le x or 6,3Ј-sulfo-Le x (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 lipidlinked oligosaccharides were used as inhibitors. These consisted of cholesterol:lecithin:lipid-linked oligosaccharides at ratios of 0.4:0.4:1 by weight.

Binding of Multivalent L-selectin to Mono-and Multisulfated
Sequences-In accord with earlier observations (6), the 3Јsulfo-Le x sequence was bound by human L-selectin but less strongly than 3Ј-sulfo-Le a . However, the 6Ј-sulfo-Le x was not bound (Fig. 1A). The 6-O-sulfation of 3Ј-sulfo-Le x to give 6,3Јsulfo-Le x elicited enhanced L-selectin binding, whereas the 6Ј-sulfated analog (6Ј,3Ј-sulfo-Le x ) consistently showed some diminution of binding. The binding signal observed with the 6,3Ј-sulfo-Le x sequence was of the same order as that observed with the 6-sulfo,3Ј-sialyl-Le x (Fig. 1C). Thus, 6-sulfation, but not 6Ј-sulfation, has a potentiating effect on human L-selectin binding not only to the 3Ј-sialyl-Le x , as shown previously (Ref. 10 and Fig. 1B), but also to the 3Ј-sulfo-Le x sequence. The hindering effect of the 6Ј-O-sulfation was less pronounced on 3Ј-sulfo-Le x than on the 3Ј-sialyl-Le x analog investigated previously (Fig. 1B) (10).
Binding experiments with the oligosaccharide C4U and its 3Ј-fucosylated analog, Fuc-C4U, clearly showed that the presence of the fucose residue is essential for L-selectin binding to the 3Ј-sialyl,6-sulfated di-N-acetyllactosamine backbone (Fig.  1D). The binding signals with the Fuc-C4U and the 6-sulfo,3Јsialyl-Le x were similar, indicating that the additional sulfates on the internal galactose and N-acetylglucosamine residues do not influence the L-selectin binding signal. Inhibition of the Binding of Multivalent L-selectin-Inhibition experiments were performed with the multivalent L-selectin using as immobilized ligands the 6-sulfo,3Ј-sialyl-Le x or the 6,3Ј-sulfo-Le x (Figs. 2 and 3, A and B); all of the acidic compounds of the Le x series tested gave some inhibition of binding. The nonsulfated lacto-N-tetraose gave no inhibition. The 6,3Јsulfo-Le x (IC 50 1.4 ϫ 10 Ϫ7 and 1.5 ϫ 10 Ϫ7 M with the two immobilized ligands) was approximately 3 and 5 times more active as an inhibitor than the 6-sulfo,3Ј-sialyl-Le x (5.1 ϫ 10 Ϫ7 and 6.9 ϫ 10 Ϫ7 M). The least active inhibitors were the nonsulfated 3Ј-sialyl-Le x (IC 50 3.7 ϫ 10 Ϫ6 and 6.8 ϫ 10 Ϫ6 M), the 6Ј-sulfo-Le x (3.1 ϫ 10 Ϫ6 and 2.0 ϫ 10 Ϫ6 M) and the 6Ј-sulfo,3Јsialyl-Le x (7.4 ϫ 10 Ϫ6 and 1.0 ϫ 10 Ϫ6 M). 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. 2B.
Inhibition of the Binding of Paucivalent L-selectin-Inhibition of binding experiments using the paucivalent L-selectin was performed (Figs. 3, C and D, and 4) to discriminate more clearly (6) 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 6,3Јsulfo-Le x was harder to inhibit than to the 6-sulfo,3Ј-sialyl-Le x (Fig. 3, C and D). Here the 3Ј-sialyl-Le x series were poorer inhibitors overall than the corresponding 3Ј-sulfo-Le x analogs and gave no significant inhibition when the 6,3Ј-sulfo-Le x was used as the immobilized ligand (Fig. 3D); when the 6-sulfo,3Ј-sialyl-Le x was used as the immobilized ligand (Fig. 3C), only the homologous oligosaccha- Varying amounts of (neo)glycolipids in a solution of carrier lipids were applied onto the microwells, and 50 ng of L-selectin/well, in the presence of anti-immunoglobulins, was added. Binding was detected by a biotin-streptavidin-horseradish peroxidase system as described previously (6). Results are expressed as means in duplicate wells with the range indicated by error bars. The results in B are taken from Ref. 10. See Table I Table I for abbreviations. ride sequence showed reasonable inhibition (IC 50 1.6 ϫ 10 Ϫ6 M). In contrast, among the oligosaccharides in which there was 3Ј-sulfate instead of 3Ј-sialyl, all inhibited the selectin binding to the two immobilized ligands. The 6Ј-sulfo-Le x was not inhibitory. The 3Ј-sulfo-Le x and the 6,3Ј-sulfo-Le x stood out as superior inhibitors with IC 50 values of 1.4 ϫ 10 Ϫ7 and 5.0 ϫ 10 Ϫ7 M, and 5 ϫ 10 Ϫ8 and 2.7 ϫ 10 Ϫ7 M, respectively. Also included in the present inhibition experiments for comparison was the 3Ј-sulfo-Le a sequence; its potency was intermediate between that of 3Ј-sulfo-Le x and 6,3Ј-sulfo-Le x (Fig. 3, C and D). DISCUSSION It is clear from the binding experiments described here, first, that, in contrast to 3Ј-sulfation of Le x , which supports L-selectin reactivity, the 6Ј-sulfation does not elicit a detectable binding signal. Second, the L-selectin reactivities of 3Ј-sulfo-and 3Ј-sialyl-Le x are similarly influenced by the addition of 6Јsulfate or 6-sulfate. Whereas 6Ј-sulfation has a negative effect, 6-sulfation has an enhancing effect on L-selectin binding to the clustered, immobilized 3Ј-sulfo-and 3Ј-sialyl-Le x series. Thus the 6-sulfate seems to be a part of the recognition motif for L-selectin, whereas 6Ј-sulfate is not. Moreover, its presence partially hinders the recognition of the 3Ј-sialyl-or 3Ј-sulfo-Le x by the selectin. The enhancement of L-selectin binding in the presence of 6-sulfation was substantial for both the 3Ј-sulfo-and the 3Ј-sialyl-Le x sequences, but the negative effect of 6Јsulfation was less pronounced with the 3Ј-sulfo-Le x than with the 3Ј-sialyl-Le x sequence investigated previously (10). Third, whereas the 6-sulfate on the subterminal N-acetylglucosamine of the Le x 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 3Ј-linked fucose on 6-sulfo,3Ј-sialyl-Le x is essential for Lselectin binding. In accord with this finding is an earlier report that L-selectin binding is abolished by modification of the fucose in the 3Ј-sialyl-Le x sequence by removal of oxygen at position 2, 3, or 5 (18). The presence of the fucose is apparently less critical in the 3Ј-sulfo-Le x sequence as it has been observed that the defucosylated 3Ј-sulfo-Le x tri-and tetrasaccharides are bound by L-selectin, albeit less strongly than the 3Ј-fucosyl analogs (4,5).
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 3Ј-sulfo than the 3Ј-sialyl-Le x . The 6-sulfated 3Јsulfo-Le x sequence had a greater inhibitory activity than the 6-sulfated 3Ј-sialyl-Le x , although the binding signals they elicited were similar. The differences were more apparent in inhibition experiments where the paucivalent L-selectin was used;  (Fig. 4, taken from experiments 4 and 6), the IC 50 values were normalized relative to that of 6,3Ј-sulfo-Le x , which was included as a reference compound. See Table I for abbreviations. 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 3Ј-sulfo-Le x series were greater than those of the 3Ј-sialyl-Le x series. Collectively, these data, together with the earlier finding (4) that the 3Ј-sulfo-but not the 3Ј-sialyl-lactosamine backbone (in the absence of fucose) elicits a binding signal with L-selectin, indicate that the 3Ј-sulfation at the galactose of Le x creates a higher affinity ligand than the 3Ј-sialylation and that L-selectin has overall a higher affinity toward the 3Ј-sulfo-and 6,3Ј-sulfo-Le x sequences than the corresponding 3Ј-sialyl-Le x 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 (6); the IC 50 values are in the nanomolar range (30 -50 nM with the best inhibitors) when displayed on liposomes and millimolar with the free oligosaccharides. This is in accord with results from other groups who have examined free oligosaccharides as inhibitors of L-selectin binding; the reported IC 50 values range from 0.2 to 6 mM (19 -22). When examined in the oligomeric state on dendrimers, IC 50 values in the 1-10 M range were reported (23). 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 Le x and Le a 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 3Ј-sulfo-Le x series and also the 3Ј-sulfo-Le a are more effective inhibitors of the L-selectin binding than the 3Ј-sialyl-Le x analogs. Among these, the 6,3Ј-sulfo-Le x and the monosulfated 3Ј-sulfo-Le a 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-Le x and -Le a would seem the most appropriate.