Sialic acid (Sia) residues of N-linked sugar chains on blood and vascular proteins are usually attached in alpha2-6 or alpha2-3 linkages, and some molecules are selectively O-acetylated at the 9- position. This diversity can affect recognition of Sia by certain vascular lectins in vitro. We will elucidate the in vivo biological significance of these findings by manipulation of certain genes in the mouse. Sialic acid O-acetyltransferase(s) have been difficult to purify or clone. We have isolated cDNA and genomic clones of a mouse esterase (Lse) that selectively removes 9-O-acetyl groups from Sias. Expression of Lse is highly regulated e.g., in murine hematopoietic development. 9-O-acetylation also occurs preferentially on the sequence Siaalpha2-6Galbeta1-4GlcNAc, whose expression is determined by the alpha2-6 sialyltransferase gene (ST6Gal I). This gene is regulated by differential action of multiple promoters in cells such as hepatocytes, endothelial cells, erythrocytes and lymphocytes. Thus, alterations in activity of ST6Gal I and/or the competing alpha2-3 sialyltransferases (ST3Gal III & IV) may also alter 9-O-acetylation. To elucidate the in vivo regulation of 9-O-acetylation and to pursue specific hypotheses about its roles,we will do the following: 1. Eliminate expression of the Lse gene systemically, and/or selectively in the cell types listed above. 2. Alter ratios of alpha2-6 and alpha2-3 Sia by disrupting expression of alpha2-6 and alpha2-3 sialyltransferases (Collaboration with Project 1) and by """"""""knocking in"""""""" the cDNA for ST3Gal III into the endogenous ST6Gal I gene, disrupting the latter. In the latter mice, alpha2-6 and alpha2-3 Sia linkage ratios should be inverted on N-glycans. 3. Elucidate the changes in 9-O- acetylation occurring in these mice, and structural and functional consequences of these genetic manipulations. Changes will be detected in situ by immunohistology using specific probes for sialylation and O-acetylation, and directly demonstrated by chemical analyses. Particular attention will be directed towards hepatocytes, endothelial cells, erythrocytes and lymphocytes. Biological effects upon interactions involving the Sia-binding lectins H protein, CD22, CD33 and Sialoadhesin will be studied (some in collaboration with Projects 1 and 3). The sensitivity of red cells to complement and the turnover of red cells and plasma proteins will also be assessed. Selected aspects of blood coagulation, hematopoiesis, and the distribution and function of lymphocytes will also be examined. These studies will help to define the in vivo regulation and biological roles of Sia O-acetylation in the blood and vascular systems.
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