The objective of this project is to define the biochemical and biophysical mechanisms through which the selectins initiate rolling of leukocytes on the vessel wall under shear forces. Rolling is a prerequisite for stable adhesion and, ultimately, for emigration of leukocytes into the tissues in response to infection or injury. The work will focus on the interactions of P- and E-selectin, which are expressed on activated platelets and/or endothelial cells, with PSGL-l, a mucin- like glycoprotein expressed on leukocytes. We have shown that these interactions are critical for neutrophil rolling on P- or E-selectin. We have purified native or recombinant forms of PSGL-l and the selectins, and have developed monoclonal antibodies to each molecule. We will determine whether the consensus repeats and EGF domains of E and P- selectin affect their kinetics and affinities of binding to PSGL-l, and we will explore the structural basis for high affinity binding of Ca2+ to the lectin domains of the selectins. We will map the binding sites for mAbs and for the selectins on PSGL- 1, and seek to express a recombinant PSGL-1 that is glycosylated such its function resembles that of the native myeloid cell glycoprotein. We will examine the structural requirements for E- and P-selectin to mediate rolling of myeloid cells under shear stresses. Finally, we will examine the mechanical strength of binding of purified PSGL-1 incorporated into liposomes to P-selectin incorporated into planar membranes, under shear forces or using micromanipulation techniques. This project is closely linked with Project 4, where the structures of the glycans of PSGL-I will be determined. Within the context of the program project, this work is designed to extend our knowledge of protein-carbohydrate interactions, biomechanical factors affecting cell-cell adhesion under shear stress, and the recruitment of inflammatory cells to the vessel wall.
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