Inflammatory diseases contribute to morbidity and mortality and are distinguished by rapid recruitment of inflammatory cells. This recruitment requires the combined actions of cytokines, chemoattractants, and adhesion receptors and ligands. P-, L- and E-selectins mediate leukocyte rolling and are required for the recruitment of inflammatory cells in vivo. PSGL-1 is a dimeric mucin (approximately 120 kD subunits) on human and murine leukocytes that is a high affinity ligand for P- and L-selectins. P- and L-selectin appears to bind to PSGL-1 through recognition of its extreme N-terminus containing sulfated tyrosine residues (TyrSO3) and core-2 based O-glycans expressing the sialyl Lewis x antigen (C2-O-sLe-x). We have developed a novel approach using semi-synthetic glycosulfopeptides (GSPs) to define the molecular nature of these interactions. Preliminary results suggest that P- selectin binds to PSGL-1 in a stereospecific manner involving distinct contributions within PSGL-1 of the three TyrSO3 residues, peptide determinants, and fucose/sialic acid on an optimally positioned C2-O- sLe-x. To explore this hypothesis and define the unusual nature of this complex interaction and its physiological significance, three specific aims are proposed. (1) We will synthesize a wide panel of GSPs with altered primary sequences based on the human and murine PSGL-1 N-termini and with specific placements of TyrSO3 residues and both core- 1 and core-2 based O-glycans. We will complete studies on the structures of the N- terminal tryptic fragment of human leukocyte PSGL-1. (2) We will define the binding of GSPs to human and murine P-, E- and L-selectins using cell adhesion/inhibition studies in vivo and in vitro, equilibrium binding studies, NMR and co-crystallization/X-ray diffraction. (3) Mice serve as the experimental model for exploring ligand/selectin interactions in vivo, but nothing is known about the nature of PSGL-1 in mouse leukocytes. We will define the structures of O- and N-glycans and sulfated moieties in PSGL-1 from murine leukocytic WEHI-3 cells that bind murine P- and E-selectin. Novel murine-based GSPs will be generated from this information and their interactions with murine P-, E- and L-selectin will be defined Our long-term goal is to understand the molecular nature of protein- carbohydrate interactions that regulate selectin functions. Such an understanding will directly contribute to the control and treatment of inflammatory diseases and should have broad implications for other types of glycobiological interactions.
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