LIPID-LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

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N-linked glycoproteins are important receptors in many eucaryotic cells, and these types of molecules have been implicated in various recognition phenomena including cell-cell interactions, host-parasite and host-symbiote interactions, and the binding of many biologically-important modlecules. Thus, these types of glycoproteins may be of especial significance in such disease conditions as cancer, atherosclerosis and diabetis. The major objective of this proposal is to understand the mechanism of biosynthesis and processing of the oligosaccharide portion of these N-linked glycoproteins. The approach to be taken in these studies is twofold. In the first case, we will solubilize as many of the glycosyl transferases and processing glycosidases as possible, purify them to some extent, and study their properties. We will be particularly concerned with the specificity with regard to substrate utilized and to the nature of the product formed in each reaction. In preliminary studies, we have already found that some of the activity for synthesizing the lipid-linked oligosaccharides can be solubilized by treating soybean cell membranes with NP-40. We also have lipid-linked oligosaccharides to use as acceptors with these enzymes so that we can assay them in soluble form. Thus we are ready to proceed with some purification studies. We have also detected glucosidase and mannosidase activities in the solubilized fraction and can assay these enzymes with oligosaccharide substrates that we have available. So we should also be able to proceed with purification here. These enzymes will be of special interest with regard to substrate specificity of the oligosaccharides utilized. In the second type of approach, we will extract various wild plants that grow in Texas, and are known to be toxic for livestock, to look for inhibitors of glycoprotein processing. This idea is based on the finding that swainsonine, an inhibitor of lysosomal Alpha-mannosidase and glycoprotein processing, is found in several toxic plants, i.e., the australian plant Swainsona canescens and american locoweed. Since swainsonine has 3 assymetric hydroxyl groups, it seems likely that other species of locoweed, or other plants, will have stereoisomers of swainsonine which may inhibit other glycosidases.