GLYCOPROTEIN HORMONE OLIGOSACCHARIDES

这笔赠款的长期目标是确定一个独特的N连锁家族的生物学意义 含有GalNAcβ1，4GlcNAcβ1，2Man-序列的碳水化合物结构，而不是序列 半乳糖β1，4GlcNAcβ1，2Man&存在于许多糖蛋白中。β-1，4-N-乙酰半乳糖基转移酶 将GalNAc添加到这个结构中的(BetaGts)是蛋白质选择性的，识别特定的氨基酸序列 被修饰的一组独特的糖蛋白。当存在于底物蛋白质中时， 识别序列可以提高GalNAc转移到碳水化合物受体500的催化效率 收牌。末端GalNAc可以保持不变或被SO4取代以形成末端SO4-4-GalNAc或 与唾液酸(SIA)形成SIA&2，6GalNAc。我们已经证明了糖蛋白，如促黄体激素 (Lh)甘露糖/GalNAc-4-SO4-受体从血液中清除含有SO4-4-GalNAc的物质，而那些 携带GalNAc或SIA的2，6GalNAc被去唾液酸糖蛋白受体去除。使用遗传 改变黄体生成素上碳水化合物结构的策略，我们已经证明了 碳水化合物决定血液中的黄体生成素浓度以及雌激素和睾丸素的含量。 被生产出来的。结果，性发育和生殖行为都发生了改变。我们会 表征糖蛋白底物中的识别决定簇，该识别决定簇通过一种新的新的 测试系统，使我们能够确定在体内和体外添加GalNAc的效率。我们会 还要检查不同的结构域或区域的蛋白质选择性BetaGT如何有助于选择性 将GalNAc加到黄体生成素和其他糖蛋白上。GalNAc家族的成员，包含有 我们在黄体生成素和其他脑垂体糖蛋白上也发现了糖蛋白产生的糖蛋白。 脑、肾、唾液腺和其他组织。我们将定义GalNAc-4基因消融的影响- 磺基转移酶和蛋白选择性β-GTs对促黄体生成素功能的影响。因为我们已经证明了烧蚀 GalNAc-4-磺酸转移酶对生殖有影响，我们预计，消融其中一个或两个β-GT是 对蛋白质的选择性将产生更大的影响，并揭示这些碳水化合物的额外功能。我们 也将定义调节转移酶的表达，这些转移酶介导这一家族的合成 结构。它们的表达可能会在激素周期的过程中以及在特定的时间段发生变化 性发育，如性成熟时。我们已经清楚地表明，改变 黄体生成素上碳水化合物的结构对其体内功能有重要影响。我们已经证明了 糖基化模式在体内是受调节的。这是第一批调制的情况之一 碳水化合物结构已被证明在体内有影响。准确定义这一点是如何监管的 对于理解生殖生物学和了解糖基化在调节细胞周期中的作用至关重要 多种糖蛋白的性质，如激素、受体和基质成分。

The long term objective of this grant is to define the biological significance of a family of unique N-linked carbohydrate structures that contain the sequence GalNAc beta1,4GlcNAc beta1,2Manα-, rather than the sequence Gal beta1,4GlcNAc beta1,2Manα- that is found on many glycoproteins. The beta1,4-N-acetylgalactosaminyl-transferases ( betaGTs) that add GalNAc to this structure are protein-selective, recognizing specific amino acid sequences in the distinctive set of glycoproteins that become modified. When present in the substrate protein, the recognition sequences can increase the catalytic efficiency of GalNAc transfer to a carbohydrate acceptor 500 fold. The terminal GalNAc can remain unmodified or be substituted with SO4 to form terminal SO4-4-GalNAc or with Sialic acid (Sia) to form Siaα2,6GalNAc. We have shown that glycoproteins such as luteinizing hormone (LH) that bear SO4-4-GalNAc are removed from the blood by the Mannose/GalNAc-4-SO4-receptor while those bearing either GalNAc or Siaα2,6GalNAc are removed by the asialoglycoprotein-receptor. Using genetic strategies to alter the structure of the carbohydrates on LH, we have demonstrated that the precise structure of the carbohydrates determine the LH concentration in the blood and the amount of estrogen and testosterone that are produced. As a result sexual development and reproductive behavior are altered. We will characterize the recognition determinant in glycoprotein substrates that is utilized by the betaGTs via a novel new assay system that allows us to determine the efficiency of GalNAc addition both in vivo and in vitro. We will also examine how different domains or regions of the protein-selective betaGTs contribute to the selective addition of GalNAc to LH and other glycoproteins. Members of the family of GalNAc containing structures that we have characterized on LH and other pituitary glycoproteins are also found on glycoproteins produced in the brain, kidney, salivary glands, and other tissues. We will define the impact of genetic ablation of the GalNAc-4- sulfotransferases and the protein-selective betaGTs on LH function. Since we have shown that ablation of GalNAc-4-sulfotransferase has effect on reproduction, we expect that ablating one or both of the betaGTs that are protein-selective will have an even greater effect and reveal additional functions for these carbohydrates. We will also define regulation of the expression of the transferases that mediate the synthesis of this family of structures. Their expression may change over the course of the hormonal cycle and at specific times during sexual development such as when sexual maturity is attained. We have clearly shown that changing the structures of the carbohydrates on LH has a significant impact on its in vivo function. We have demonstrated that the pattern of glycosylation is modulated in vivo. This is one of the first instances in which modulation of carbohydrate structures has been shown to have an impact in vivo. Defining precisely how this is regulated is critical for understanding reproductive biology and for understanding the role of glycosylation in modulating the properties of a wide range of glycoproteins such as hormones, receptors, and matrix components.