Enzymes that transfer sialic acids to glycoproteins and glycolipids are termed sialyltransferases. Sialic acid addition generally terminates further oligosaccharide biosynthesis and hence can generate or mask biologically relevant oligosaccharide structures. Among vertebrates, a highly conserved sialyltransferase gene family exists that contains over a dozen members. These genes exhibit expression profiles that are commonly developmentally restricted and cell- or tissue-type specific. From limited yet provocative studies, sialic acid linkages have been found to regulate cellular interactions involving trafficking, adhesion, and signal transduction, as in the mechanism of selectin function. Additionally, sialic acid linkages may control the stability and activity of various glycoproteins in the plasma. The overall goal of this project is to understand the in vivo biological significance of sialyltransferase-generated alpha2-3 and alpha2-6 sialic acid linkages. Their expression is differentially regulated in cells of the hematopoietic lineage (Projects 2 and 3), and well as in the endothelium and liver (sources of most of the coagulation factors). Moreover, alpha2-6 sialyltransferase gene expression is normally induced in the liver and endothelium during inflammatory responses. The resulting sialic acid linkages are recognized by the newly identified family of I-type lectins on hematopoietic cells that include CD33 and sialoadhesin (Project 3) and may be biologically regulated by O-acetylation (Project 4). To study sialyltransferase function in vivo, this proposal includes the production and analysis of systemic null mutations, as well as tissue-and cell-specific null mutations using Cre-loxP site-directed recombination. This will allow studies of the roles of these sialyltransferases and the resulting oligosaccharides in specific physiologic systems likely affected, involving hepatocytes, vascular endothelium, and hematopoietic cell types. Phenotypes will be studied using lectin binding, immunohistology, oligosaccharide structural analysis, and in situ hybridization. Effects on hematopoiesis, blood coagulation, inflammatory responses, and immunologic competence will be assessed. Phenotypes obtained may reflect the exposure of galactose residues on oligosaccharide termini, their modification by other sialyltransferases, and possible variations in sialic acid O- acetylation profiles. Results of these studies are hypothesized to include alterations in cell trafficking and adhesion, protein stability, and perhaps signal transduction, thereby disclosing biological systems that require specific sialic acid linkages in regulating normal and possibly disease physiology.
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