MOLECULAR BASIS OF SELECTIN INTERACTIONS WITH LEUKOCYTES

Oklahoma Medical Research Foundation, Oklahoma City OK

Investigators

Linked publications & trials

Abstract

In shear flow, leukocytes use the mucin PSGL-1 to tether to and roll on P-selectin on activated platelets and endothelial cells and on L-selectin on adherent leukocytes. P-and L-selectin bind to a small N-terminal region of PSGL-1 that requires both tyrosine sulfate (TyrSO3) and a core-2 O-glycan capped with sialyl Lewis x (C2-0-sLeX). A synthetic glycosulfopeptide modeled after this region binds with the same affinity as native PSGL-1 to P-selectin. The investigator will use four approaches to determine the biochemical and biophysical features that enable selectin-PSGL-1 interactions to mediate cell adhesion in shear flow: (1) Investigator will compare the affinity, kinetics, and thermodynamics of binding of soluble monomers of P-, L-, and E-selectin to native PSGL-1 and recombinant soluble PSGL-1 and to synthetic glycosulfopeptides modeled after the N terminus of PSGL-1. Binding will be measured by surface plasmon resonance, fluorescence, and microcalorimetry. (2) The investigator will use Xray crystallography to determine the three-dimensional structures of the lectin and EGF domains of P-selectin and L-selectin, alone or complexed with a glycosulfopeptide modeled after the N terminus of PSGL-1. The structural information will be used to test contributions of specific amino acids to the ligand-binding characteristics of each selectin. (3) The investigator will determine whether PSGL-1 on some cells uses posttranslational modifications other than TyrSO3 and C2-0-sLeX to confer selectin binding. (4) The investigator will examine the kinetic and mechanical properties of selectin-PSGL-1 bonds that mediate tethering and rolling adhesion of cells in shear flow. The investigator will study interactions of cells expressing wild-type or altered selectins and PSGL-1, and the investigator will perfuse microspheres coupled with glycosulfopeptides modeled after PSGL-1 over immobilized selectins A key component will be coupling of flow chamber data to mathematical models, which will allow evalluation of physiochemical properties and prediction of adhesiive behaviors. These studies will provide new insigrhts into the mechanisms by which selectins initiate leukocyi:e adhesion in shear flow.