Inhibition of L-selectin-mediated Leukocyte Rolling by Synthetic Glycoprotein Mimics

Abstract

Synthetic carbohydrate and glycoprotein mimics displaying sulfated saccharide residues have been assayed for their L-selectin inhibitory properties under static and flow conditions. Polymers displaying the L-selectin recognition epitopes 3,6-disulfo Lewis x(Glc) (3-O-SO 3 -Gal␤1␣4(Fuc␣1␣3)-6-O-SO 3 -Glc␤-OR) and 3,6-disulfo Lewis x(Glc) (3,6-di-O-SO 3 -Gal␤1␣4(Fuc␣1␣3)Glc␤-OR) both inhibit L-selectin binding to heparin under static, cell-free binding conditions with similar efficacies. Under conditions of shear flow, however, only the polymer displaying 3,6-disulfo Lewis x(Glc) inhibits the rolling of L-selectin-transfected cells on the glycoprotein ligand GlyCAM-1. Although it has been shown to more effective than sialyl Lewis x at blocking the L-selectin-GlyCAM-1 interaction in static binding studies, the corresponding monomer had no effect in the dynamic assay. These data indicate that multivalent ligands are far more effective inhibitors of L-selectin-mediated rolling than their monovalent counterparts and that the inhibitory activities are dependent on the specific sulfation pattern of the recognition epitope. Importantly, our results indicate the L-selectin specificity for one ligand over another found in static, cell-free binding assays is not necessarily retained under the conditions of shear flow. The results suggest that monovalent or polyvalent carbohydrate or glycoprotein mimetics that inhibit selectin binding in static assays may not block the more physiologically relevant process of selectin-mediated rolling.

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…Physiological L-selectin ligands typically present multiple copies of derivatives of the sulfated sialyl Lewis × antigen (sLe × ); synthetic multivalent ligands that display these and related carbohydrate epitopes have been shown to be effective selectin ligands. [248][249][250][251][252][253] Multivalency, therefore, may be an important determinant of L-selectin function in vivo. Experiments using synthetic multivalent ligands have begun to reveal the importance of multivalency and stoichiometry of selectin-ligand clusters for L-selectin recognition.…”

Minimal Sulfated Carbohydrates for Recognition by L-selectin and the MECA-79 Antibody

“…Given that L-selectin functions in the dynamic processes of tethering and rolling of lymphocytes, a complete analysis of 6-sulfo-sLe X must examine the contribution of each modification (including sulfation) to kinetic constants as well as to the overall equilibrium constant. A previous study has shown that sulfated Le X derivatives can exhibit very different inhibitory activities against L-selectin depending upon whether equilibrium or flow chamber assays are used (37).…”

“…Furthermore, Kannagi and coworkers (33, 34) have described 6-sulfo-sLe X -reactive antibodies that stain HEVs and inhibit L-selectin binding to HEVs. With respect to the contribution of Gal-6-SO 4 , several groups have reported that this modification of sLe X or of sLe X mimetics either enhances or does not affect L-selectin binding (29,30,(35)(36)(37). In marked contrast, Feizi and co-workers found that this modification eliminates binding to L-selectin (28, 38).…”

Specific Binding of Glucose-derivatized Polymers to the Asialoglycoprotein Receptor of Mouse Primary Hepatocytes

“…The second combined effect is the polymeric effect of the PV6Gna engaging ASGPR in multivalent binding on the hepatocyte cell surface. Multivalent synthetic glycoprotein inhibits L-selectin-mediated leukocyte rolling more effectively than the corresponding monomer (7). Several reports have demonstrated a multivalent effect of ligands for an optimum recognition to the structure of ASGPRs (49,50).…”

“…Multivalent glycopolymer ligands have been designed for clustering of L-selectin leading to the activation of the leukocyte cell surface and the inhibition of L-selectin-mediated leukocyte rolling (6,7). Interaction between the carbohydrate and the carbohydrate-binding proteins (CBPs) 1 is achieved through hydrogen bonding of the hydroxyl group of the carbohydrate to the polar amino acid side chain of the protein, including the packing of a hydrophobic sugar face against the aromatic amino acid side chain of the protein (8).…”