In recent years, specific biological functions have been attributed to modifications of the oligosaccharide units of glycoconjugates, such as phosphorylation, O-acetylation and sulfation. The long term aimn of this program is to study modifications of N-linked oligosaccharides and to understand their biological roles in helath and disease. We have discovered two novel classes of sulfated N- linked oligosaccharides in mammalian cells. The first is a class of multiantennary complex-type oligosaccharides carrying Sialic acids and O-Sulfate esters (SSOs) and the second is a family of N- Linked molecules carrying Glycosaminoglycan-like chains attached to complex-type oligosaccharides (NLGs). This application proposes to study the complete structures and biosynthesis of these novel molecules, and to explore their biological roles. The complete structures will be elucidated using both labelled and unlabelled matrials, by the production of monoclonal antibodies against unique epitopes, and by the purification of specific sulfatases. particular attention will be paid to the nature of the linkage between the sulfated side chains and the core portion of the oligosaccharide. In selected cases, the nature of the proteins carrying these molecules will also be investigated. The effects of para-nitrophenyl xyloside and of selected inhibitors of processing glycosidases upon the biosynthesis of both types of molecules will be explored. The biosynthetic intermediates of these molecules will be identified by labelling either intact Golgi vesicles, cells at lowered temperature, or cells exposed to monensin. This will allow the prediction of the overall biosynthetic pathways for the initiation and synthesis of both types of chains. Based upon all of the above, selected studies of the enzymology of these pathways will be carried out. In parallel with these studies, we will examine further the structure and biosynthesis of both SSOs and NLGs that we have found on a single pancreatic tumor antigen. The unusual nature of these structures suggests that they have interesting biological roles. Labelled SSOs will be used as ligands to search for their cognate receptors in cell membranes. Preliminary studies suggest that the LGs of pulmonary vascular endothelial cells bind to Antithrombin III, and therefore, could have a role in the control of thrombogenesis in the pulmonary vasculature. We will also compare the biological roles of the NLGs with others already known for the more typical O-linked heparin/heparan sulfate proteoglycans. Other biological roles for both types of structures may be predicted by the structural, biosynthetic and receptor binding studies. in the long run, studies of these novel molecules may be of importance in such diverse processes as the control of endothelial and smooth muscle proliferation, the adhesion of tumor cells in the process of metastases, and the control of thrombogenesis.