STRUCTURE-FUNCTION ANALYSIS OF P-SELECTIN-SIALYL LEWIS(X) BINDING INTERACTIONS - MUTAGENIC ALTERATION OF LIGAND-BINDING SPECIFICITY

P-selectin is a vascular cell adhesion molecule that is expressed on t he surface of platelets and endothelial cells in response to inflammat ory stimuli. It is believed to aid in the binding and recruitment of l eukocytes to inflamed tissue. P-selectin adhesion to leukocytes is med iated by the amino-terminal lectin domain that binds the sialyl Lewis( X) (sLe(X)) carbohydrate (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)G lcNAc). Neither the three-dimensional structure of P-selectin nor the protein-carbohydrate interactions that mediate the binding of P-select in to the sLe(X) carbohydrate have been determined. The most closely r elated protein for which a ligand-bound three-dimensional structure ha s been resolved is the rat mannose-binding protein (Weis, W. L., Drick amer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134). Using th e known binding interactions that occur between the rat mannose-bindin g protein and its ligand (oligomannose) as a template, we have used si te-directed mutagenesis to substitute Ala-77 with lysine. This substit ution changed P-selectin-carbohydrate binding specificity from sLe(X) to oligomannose. Further substitution altered the binding preference f rom mannose to galactose in a predictable manner. These results indica te that P-selectin binds sLe(X) 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 resi due that has previously been implicated in P-selectin binding to both sLe(X) and 3-sulfated galactosylceramide (sulfatide). Our analysis dem onstrates that Lys-113 is probably not involved in P-selectin binding to either sulfatide or sLe(X). Functionally, it appears that P-selecti n 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 .