Structure-function analysis of P-selectin-sialyl LewisX binding interactions. Mutagenic alteration of ligand binding specificity.

P-selectin is a vascular cell adhesion molecule that is expressed on the surface of platelets and endothelial cells in response to inflammatory stimuli. It is believed to aid in the binding and recruitment of leukocytes to inflamed tissue. P-selectin adhesion to leukocytes is mediated by the amino-terminal lectin domain that binds the sialyl LewisX (sLeX) carbohydrate (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc). Neither the three-dimensional structure of P-selectin nor the protein-carbohydrate interactions that mediate the binding of P-selectin to the sLeX carbohydrate have been determined. The most closely related protein for which a ligand-bound three-dimensional structure has been resolved is the rat mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134). Using the known binding interactions that occur between the rat mannose-binding protein and its ligand (oligomannose) as a template, we have used site-directed mutagenesis to substitute Ala-77 with lysine. This substitution changed P-selectin-carbohydrate binding specificity from sLeX to oligomannose. Further substitution altered the binding preference from mannose to galactose in a predictable manner. These results indicate that P-selectin binds sLeX in a shallow cleft that is similar to the mannose-binding protein saccharide-binding cleft. Additionally, we present an extensive mutagenic analysis of P-selectin Lys-113, a residue that has previously been implicated in P-selectin binding to both sLeX and 3-sulfated galactosylceramide (sulfatide). Our analysis demonstrates that Lys-113 is probably not involved in P-selectin binding to either sulfatide or sLeX. Functionally, it appears that P-selectin has retained a conserved carbohydrate and calcium coordination site that enables it to bind carbohydrate in a manner that is quite similar to that which has been determined for the rat mannose-binding protein.