The research outlined in this proposal is to study the biosynthesis, structure, and function of glycoproteins, particularly with respect to the carbohydrate portion of these molecules. We will examine the mechanism of biosynthesis of glycoproteins in aorta tissue. Aortic extracts incorporate the mannose moiety of GDP-C14-mannose into several glycolipids and into glycoprotein. One of these glycolipids is a mannosyl-phosphoryl-polyisoprenol whereas the other lipids appear to be oligosaccharide-polyprenols. The properties of these various "lipid-intermediates" and their mechanism of synthesis will be studied. These lipids will then be utilized as substrates with aortic extracts to demonstrate that they are intermediates in glycoprotein synthesis. Aortic tissue cultures and smooth muscle cells will also be tested for their ability to form "lipid-intermediates" and glycoproteins both in vivo and in vitro. We have also found that extracts of Mycobacterium smegmatis incorporate mannose from GDP-C14-mannose into mannosylphophoryl-polyprenol and into oligosaccharide-polyprenols. The utilization of these intermediates in the formation of complex carbohydrates of the cell wall of this organism will be examined and the mechanism compared to that of aorta glycoprotein synthesis. A number of glycoprotein enzymes are secreted by the fungus Aspergillus fumagatus. Two of these glycosidases, a beta-mannosidase and a beta-galactosidase, have recently been purified to homogeneity and their chemical composition and structure will be studied as well as their substrate specificity. These enzymes will be useful tools for studying the composition of mannose and galactose polymers, such as the above biosynthetic glycoproteins.