Glycoprotein Hormone Oligosaccharides

描述（由申请人提供）：该资助的长期目标是了解以序列S 04 -4-Ga 1 NAcBeta 1，4GlcNAcBeta 1，2 Manalpha终止的独特碳水化合物结构家族的生物学作用。我们以前已经证明，N-连接糖蛋白激素促黄体激素（LH）和促甲状腺激素（TSH）从所有脊椎动物物种终止这种独特的硫酸化结构。此外，我们已经表明，这些硫酸化结构控制LH的循环半衰期，并通过下丘脑-垂体-性腺轴调节雌激素的产生。我们现在已经克隆了两种GalNAc-4-磺基转移酶，GaINAc-4-ST 1和GaINAc-4-ST 2，其将硫酸盐转移到末端β 1，4-连接的GaINAc。GaINAc-4-ST 1在垂体中高度表达，并解释了LH和TSH的N-连接寡糖上存在末端GalNAc-4-S 04的原因。由脑、肾、脾、淋巴结、卵巢、睾丸和子宫中的细胞产生的以β 1，4-连接的GaINAc-4-S 〇 4终止的另外的糖蛋白表明这些结构对于其他生物过程也将是关键的。我们将：1）表征GaINAc-4-磺基转移酶在成体和发育中的胚胎中的表达。2)生成并表征GalNAc-4-磺基转移酶缺陷小鼠。3)定义GalNAc-4-磺基转移酶的结构：功能关系。和4）克隆蛋白特异性β 1，4-GalNAc-转移酶并表征其肽识别决定簇，使用新的分泌嵌合蛋白在用cDNA表达文库转染细胞后直接筛选β 1，4-GalNAc-转移酶的表达。在整个脊椎动物进化过程中，糖蛋白激素上的这些硫酸化结构以及识别它们的受体的保守性证明了它们的重要性。遗传，生化和结构的方法相结合，我们将用来研究这些寡糖的生物学作用，将推进我们的理解，在调节雌激素产生的下丘脑-垂体-性腺轴的作用。由于LH和TSH清除率的改变，预计这些结构的合成改变会导致激素失衡。这种改变还可能导致脑发育和对抗原的免疫应答异常，这是由于在脑和淋巴结的树突细胞中具有以GaINAc-4-S 04终止的结构的糖蛋白的高度调节的表达。我们的研究还将揭示这些硫酸化寡糖在其他环境中的作用。

DESCRIPTION (provided by applicant): 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.