GLYCOPROTEIN HORMONE OLIGOSACCHARIDES

我们已经确定了一组精选的糖蛋白，包括 糖蛋白激素促黄体生成素(LH)和促甲状腺激素(TSH)，熊ASN连锁 以唯一序列SO4-4-终止的低聚糖 GalNAcbeta1，4GlcNAc-beta。这一序列是由一个肝脏识别的 内皮细胞受体，GalNAc-4-SO4-受体，迅速 将黄体生成素从循环中移除。由此产生的循环半衰期较短 对于体内表达最大的促黄体生成素生物活性是必不可少的。我们有 确定了GalNAc转移酶和负责 以GalNac-4-SO4为末端的低聚糖的合成。这些 糖基转移酶必须与多种其他转移酶竞争 完全相同的合成中间体。我们已经证明了GalNAc- 转移酶识别三肽基序ProXaArg/Lys(PXR/K) 促黄体生成素的α和β亚基，它增加了对 GalNAc-体外转移。我们将对此进行定点突变 改变酶催化效率的基序和周围序列 GalNAc体外转移。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 动力学参数的变化会影响分子的结构 在体内发现的更复杂的环境中合成的低聚糖。这个 负责合成这些硫酸盐的GalNAc-和磺基转移酶 低聚糖将被分离出来，并用两种生化方法进行表征 以及定义结构功能的分子生物学方法 两性关系。组织特异性发育和激素表达 将对GalNAc和磺基转移酶进行研究以深入了解 它们的表达调控和细胞的生物学功能 生产的硫化低聚糖。酶、生化和分子 生物研究将被用来研究这种进化。 相互依赖的转移酶和底物系统。后一种方法 将提供对生物进化发展的洞察力 对复合体中独特的低聚糖结构起作用， 多成分识别系统。

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.