The asparagine-linked oligosaccharides of bovine pituitary hormones are unique due to the presence of N-acetyl galactosamine (GalNAc) and sulfate. Sulfate is covalently linked to GlcNAc and GalNAc at the non-reducing termini of oligosaccharides on both the Alpha and Beta subunits of lutropin (LH) and thyrotropin (TSH). In contrast the human placental hormone, chorionic gonadotropin (hCG), which shares nearly identical Alpha subunit with the pituitary hormones, lacks sulfate and GalNAc. Instead it contains the terminal sialic acid on its asparagine-linked oligosaccharides. Although these comparative studies involved peptide hormones derived from different species, they suggest that some step(s) leading to the synthesis of sulfated rather than sialylated oligosaccharide on similar Alpha subunits is tissue specific. The basis of this specificity represents an important focus for this proposal. We propose to deal with the following issues: 1) Structures of sulfated and non-sulfated oligosaccharides on the intracellular and secreted hormone species synthesized in pituitary. These studies will encompass both bovine and human hormones, LH and FSH. 2) The structural and/or tissue determinants that are responsible for sulfation of these hormones. 3) Characterization of the glycosyl transferase responsible for addition of GalNAc to bLH. Experiments will be directed at determining if addition of GalNAc is tissue or protein specific and if GalNAc addition is required for sulfation. 4) Identification and isolation of the sulfotransferases. For example, are there tissue and/or substrate-specific sulfotransferases? 5) The role of sulfation in the packaging and secretion of LH. 6) The effect of sulfate on biologic function of LH including half-life in the circulation, targeting, and receptor stimulation. The model we propose will be useful for determining why particular proteins undergo certain post-translational modifications.
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