N-linked oligosaccharides terminating with the sequence S04- 4-GalNAc-beta1, 4GlcNAc-beta (SGN) have been conserved on the glycoprotein hormones of vertebrate species from fish to mammals. This implies that they serve highly important functions whose definition is a long term goal of the applicant's research. These oligosaccharide structures are recognized by an SGN-specific receptor, and play an essential role in vivo, for example, in regulating LH circulating half life to maximize its receptor activation during the ovulatory cycle. The highly regulated synthesis of these structures yields a family of oligosaccharides containing the sequence GalNAc-beta1, 4-GlcNAc-beta initiated by protein specific beta1, 4GalNAc transferase on selected glycoproteins. Three distinct family members can be produced by the subsequent addition of S04, sialic acid, or fucose by protein-independent transferases such as the GalNAc-4-sulfo transferase. This project will pursue two major specific aims: 1). A definition of the molecular basis for regulation of protein- specific and protein-independent forms of glycosylation by the beta1, 4GalNAc-T and the GalNAc-sulfoT in vitro and in vivo, and 2). Molecular definition of the structural features of the protein specific beta1, 4GalNAc transferase and the protein independent GalNAc-4-sulfoT that account for their properties and how this relates to the regulation of their expression. Relationships between the synthesis of these unique oligosaccharide structures and their biological role in vivo will be determined using constructs that introduce the protein-specific beta1, 4GalNAcT recognition determinant into another protein. Characteristic of the protein-specific beta1, 4GalNAcT and the protein independent GalNAc-4- sulfoT will be defined at molecular level, by cloning these transferases and establishing how their structural features related to their ability to selectively modify oligosaccharides. The applicant will place particular emphasis on the developmental regulation of transferase expression. Understanding the selective modification of glycoproteins with specific oligosaccharide structures will provide important information on processes central to reproductive biology, as well as malignant transformation, inflammation, cell recognition, and protein targeting.
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