Peptides Which Bind to E-selectin and Block Neutrophil Adhesion

Abstract

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with K d values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.E-selectin is a cell adhesion molecule which is induced on the surface of endothelial cells in response to inflammatory cytokines (1, 2). Binding of E-selectin to its ligand expressed on the surface of circulating neutrophils initiates rolling, an early step in the recruitment of these cells to a site of injury or inflammation (3, 4). The sequence of E-selectin (2,5,6) shows that it is a member of the mammalian C-type lectin family (7), and its three-dimensional structure shows strong similarity to mannose-binding protein (8). The carbohydrate structure sialyl Lewis x (sLe x ; 1 Neu5Ac␣2-3Gal␤1-4[Fuc␣1-3]GlcNAc) has been identified as a ligand which binds to the lectin domain at the N terminus of E-selectin (9 -12). However, natural ligands may also contain protein (13,14) or other carbohydrate structures. Inhibition of neutrophil adhesion to endothelium is an attractive approach to controlling inflammation-mediated diseases such as rheumatoid arthritis or psoriasis (15). Several potential therapeutics have been tested for their ability to inhibit the E-selectin-neutrophil adhesion event, including carbohydrate-based molecules (16, 17), antibodies (18), soluble E-selectin (19), and selectin-Ig chimeras (20). While these molecules have been useful to show the utility of selectin blockers for treating inflammation, each has significant drawbacks as a therapeutic, including short in vivo half-life, potential immunogenicity, high cost, and other possible side effects. A further limitation of these approaches is the lack of an efficient means to improve the pharmaceutical properties of these molecules.In the past few years, several methods for creating and screening vast libraries of recombinant peptides have been developed (21-24). These libraries have been used to discover novel peptide ligands for several proteins, including antibodies (21, 24, 25), receptors (26, 27), and lectins (28, 29), as well as novel enzyme substrates (30 -33). We report here the use of recombinant peptide dis...

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

“…To test this hypothesis, a peptide-displaying phage library was screened for peptides binding to E selectin (22,23). Specifically, we screened a peptide-displaying library by using an anti-Le a antibody as a target and isolated peptides that compete with sialyl Lewis x for binding to E-, L-, and P-selectin in a calcium-dependent manner (23).…”

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