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

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

doi:10.1074/jbc.274.26.18213

Abstract

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 the N-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 inner N-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.

Highlights

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]
Knowing that 3-O-sulfation at galactose of Lex or Lea 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 Lex sequences in which the 3Ј-sialyl residue on Lex is replaced by 3Ј-O-sulfate, and we make a comparison with reactivity toward the 3Ј-sulfo-Lea sequence, which is among the most potent L-selectin ligands far described, and with reactivity toward the 6Ј-sulfo-Lex
The L-selectin reactivities of 3Ј-sulfo- and 3Ј-sialyl-Lex are influenced by the addition of 6Јsulfate or 6-sulfate

Summary

Introduction

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]. The occurrence of 3Ј-sulfated forms of Lex and 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 [4, 7, 8]. Among these four sequences, the strongest binding signal is with the 3Ј-sulfo-Lea [6]. We 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

Methods

Results

Conclusion