We have established that a select group of glycoproteins including, the glycoprotein hormones lutropin (LH) and thyrotropin (TSH), bear Asn-linked oligosaccharides terminating with the unique sequence SO4-4- GalNAcbeta1,4GlcNAc-beta. This sequence is recognized by a hepatic endothelial cell receptor, the GalNAc-4-SO4-Receptor, which rapidly removes LH from the circulation. The resultant short circulatory half life is essential for the expression of maximal LH bioactivity in vivo. We have identified the GalNAc-transferase and the sulfotransferase responsible for synthesis of oligosaccharides terminating with GalNAc-4-SO4. These glycosyltransferases must compete with multiple other transferases for the identical synthetic intermediates. We have shown that the GalNAc- transferase recognizes a tripeptide motif, ProXaaArg/Lys (PXR/K), on the alpha and beta subunits of LH which increases the catalytic efficiency for GalNAc-transfer in vitro. We will use site-directed mutagenesis of this motif and surrounding sequences to alter the catalytic efficiency of GalNAc transfer in vitro. We will then express the same proteins in cells containing the GalNAc- and sulfotransferases to determine how these changes in kinetic parameters affect the structures of the oligosaccharides synthesized in the more complex milieu found in vivo. The GalNAc- and sulfotransferases responsible for synthesis of these sulfated oligosaccharides will be isolated and characterized using both biochemical and molecular biologic approaches to define structure-function relationships. The tissue specific developmental and hormonal expression of the GalNAc- and sulfotransferase will be examined to gain insight into the regulation of their expression and the biologic functions of the sulfated oligosaccharides produced. Enzymatic, biochemical, and molecular biologic studies will be used to examine the evolution of this interdependent system of transferases and substrates. The latter approach will provide insights into the evolutionary development of biologic function for unique oligosaccharide structures in a complex, multicomponent recognition system.
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