We will continue our studies on the biosynthesis and function of the N-linked oligosaccharides, using both in vivo and in vitro approaches. For in vivo studies, we will use aortic smooth muscle cells and examine the biosynthesis and function of the LDL-receptor. This receptor contains N-linked oligosaccharides and its proper function may be of critical importance in atherosclerosis. We have two plant alkaloids, swainsonine and castanospermine, that inhibit specific processing glycosidases and therefore cause dramatic alterations in the oligosaccharide structures of the glycoproteins produced in their presence. We want to know whether such alterations will affect the function of the LDL-receptor, and whether alkaloid-treated smooth muscle cells would display symptoms like those of individuals with certain types of hypercholesteremia. Another in vivo system that we will use is the influenza virus system. The viral hemagglutinin is an N-linked glycoprotein and we have previously used this system to determine the action of swainsonine and castanospermine. Since it is a very convenient system, we will use it to find other inhibitors of processing. Such compounds could be of extreme value for many functional studies related to a number of disease conditions. We will also study the biosynthesis in vitro using cell-free extracts of pig aorta to examine the reactions in the """"""""dolichol"""""""" cycle. In this case, we hope to purify some of the mannosyl transferases produce the different lipid-linked saccharide intermediates, so that we can study their properties. With some of these enzymes, and the G1cNAc-1-P transferase, we will examine the question of regulation and try to determine if and how it occurs. We will also study regulation in cell culture using inhibitors of protein synthesis or other types of inhibitors. We have some ideas for chemically synthesizing a variety of inhibitors and we have obtained a number of potential inhibitors from several pharmaceutical companies. We will test these with the particulate enzyme to determine their effect on the formation of lipid-linked saccharides. If these inhibitors work at the vitro level, we will test them in cell culture.
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