GENETIC MODULATION OF BLOOD AND VASCULAR GLYCOSYLATION

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Oligosaccharides such as the N- and 0-linked chains of glycoproteins and the heparan sulfate chains of proteoglycans are major structural components of cells and tissues, with a complexity rivaling that of nucleic acids and proteins. This Program is focused upon exploring the biological roles in the blood and vasculature of two anionic modifications of these sugar chains: sialic acid and N-sulfation. The structures of the N- and 0-linked sugar chains of many plasma glycoproteins and blood cells and are well known. Specific lectins such as the selectins (on leucocytes, platelets and endothelium), the I-type lectins (on B cells, macrophages or myeloid precursors), and the H protein of the complement pathway, can differentially recognize these chains in the context of their sialic acid residues. The N-sulfate esters of heparan sulfate glycosaminoglycan chains are recognized by various proteins involved in blood coagulation, cell growth, platelet interactions and leucocyte migration. Despite many clues, most physiologic roles of sialylation and N-sulfation are not well observed in cultured cells, and must be explored in the intact organism. However, these features of mammalian glycosylation are not well represented in model invertebrates, and few genetic defects affecting these pathways have been described in intact mammals. Our labs have been among the first to genetically dissect the in vivo roles of glycosylation in the mouse and to develop new technologies for efficient analysis of oligosaccharides from mouse tissues. Thus, the central theme of this proposal is the genetic manipulation of sialic acids and N-sulfate esters in the intact mouse. When required, we will use our recently developed method to selectively inactivate mouse genes in a cell type-specific manner. Replacement of wild type alleles with recombinant ones carrying loxP target sites at innocuous positions allows selective gene deletion, by mating with mice transgenic for Cre recombinase constructs driven by specific transcriptional control sequences. This allows specific focus on blood cells, endothelium and plasma proteins, and will be important in instances where systemic gene inactivation models are non-viable. We also have the necessary expertise to analyze the consequences of the genetic manipulations on the structure of hematopoietic, vascular and lymphoid tissues, the mechanisms of hemostasis, the structure of the oligosaccharides, and the functional consequences to hematopoiesis and lymphocyte biology. These studies are expected to reveal many important functions for oligosaccharides in health and disease.