GLYCOPROTEIN HORMONE OLIGOSACCHARIDES

我们已经确定了一组选择的糖蛋白，包括， 糖蛋白激素促黄体激素（LH）和促甲状腺激素（TSH），带有Asn连接 以独特序列SO4 - 4-终止的寡糖 GalNAc β 1，4GlcNAc-β。该序列由肝细胞识别。 内皮细胞受体，GalNAc-4-SO4-受体， 将LH从循环中去除。由此产生的短循环半衰期 是体内表达最大LH生物活性所必需的。我们有 确定了GalNAc转移酶和磺基转移酶， 以GalNAc-4-SO4终止的寡糖的合成。这些 糖基转移酶必须与多种其他转移酶竞争糖基转移。 相同的合成中间体。我们已经证明，GalNAc- 转移酶识别三肽基序，ProXaaArg/Lys（PXR/K）， LH的α和β亚基，这增加了 体外GalNAc转移。我们将利用定点突变技术 基序和周围序列，以改变催化效率 体外GalNAc转移。然后我们将在细胞中表达相同的蛋白质 含有GalNAc-和磺基转移酶，以确定这些 动力学参数的变化影响了 低聚糖在体内发现的更复杂的环境中合成。的 GalNAc-和磺基转移酶负责合成这些硫酸化 将使用生物化学和生物化学方法分离和表征低聚糖。 和分子生物学方法来定义结构-功能 关系。组织特异性发育和激素表达 的GalNAc-和磺基转移酶将进行检查，以了解 它们的表达调控和生物学功能， 产生硫酸化低聚糖。酶、生物化学和分子 生物学研究将被用来研究这种进化， 转移酶和底物的相互依赖系统。后一种方法 将为生物学的进化发展提供见解， 在复合物中具有独特的寡糖结构， 多元识别系统

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.