The long-term objective of this grant is to understand the biological role of a family of unique carbohydrate structures that terminate with the sequence S04-4-Ga1NAcBeta1,4GIcNAcBeta1,2Manalpha. We have previously demonstrated that the N-linked carbohydrates on the glycoprotein hormones lutropin (LH) and thyrotropin (TSH) from all vertebrate species terminate with this unique sulfated structure. Furthermore, we have shown that these sulfated structures control the circulatory half-life of LH and are essential for the regulation of estrogen production by the hypothalamic-pituitary-gonadal axis. We have now cloned two GalNAc-4-sulfo-transferases, GaINAc-4-ST1 and GaINAc-4-ST2, that transfer sulfate to terminal Beta1,4-linked GaINAc. GaINAc-4-ST 1 is highly expressed in the pituitary and accounts for the presence of terminal GalNAc-4-S04 on the N-linked oligosaccharides of LH and TSH. Additional glycoproteins terminating with Beta1,4-linked GaINAc-4-S04 produced by cells in the brain, kidney, spleen, lymph nodes, ovary, testis, and uterus indicate that these structures will be critical for other biological processes as well. We will: 1) Characterize the expression of GaINAc-4-sulfotransferase in the adult and developing embryo. 2) Generate and characterize GalNAc-4-sulfotransferase deficient mice. 3) define the structure:function relationships of the GalNAc-4-sulfotransferases. And 4) Clone the protein-specific Beta 1,4-GalNAc-transferase and characterize its peptide recognition determinant, using a novel secreted chimeric protein to directly screen for expression of the Beta 1 ,4-GalNAc-transferase following transfection of cells with a cDNA expression library. The conservation of these sulfated structures on the glycoprotein hormones throughout vertebrate evolution, as well as the receptor that recognizes them, attests to their importance. The combination of genetic, biochemical, and structural approaches we will use to study the biological role of these oligosaccharides will advance our understanding of their role in the regulation of estrogen production by the hypothalamic-pituitary-gonadal axis. Alterations in the synthesis of these structures are expected to lead to hormonal imbalances due to altered clearance of LH and TSH. Such alterations may also result in abnormalities in brain development and in the immune response to antigens due to the highly regulated expression of glycoproteins bearing structures terminating with GaINAc-4-S04 in the brain and dendritic cells of the lymph node. Our studies will also reveal the role of these sulfated oligosaccharides in other settings.
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