Glycoprotein Hormone Oligosaccharides

描述（由申请人提供）：本资助的长期目标是了解以S04-4-Ga1NAcBeta1,4GIcNAcBeta1,2Manalpha序列终止的独特碳水化合物结构家族的生物学作用。我们之前已经证明，所有脊椎动物的糖蛋白激素lutropin （LH）和thyrotropin （TSH）上的n -连接碳水化合物终止于这种独特的硫酸化结构。此外，我们已经证明，这些硫酸盐结构控制LH的循环半衰期，并对下丘脑-垂体-性腺轴调节雌激素的产生至关重要。我们现在克隆了两个galac -4-磺酸转移酶，GaINAc-4- st1和GaINAc-4- st2，它们将硫酸盐转移到末端Beta1,4-连接的GaINAc。GaINAc-4-ST 1在垂体中高表达，并在LH和TSH的N-linked寡糖上存在末端galac -4- s04。脑、肾、脾、淋巴结、卵巢、睾丸和子宫细胞中产生的以Beta1,4-linked GaINAc-4-S04终止的其他糖蛋白表明，这些结构对其他生物过程也至关重要。我们将：1)表征gainac -4-硫转移酶在成人和发育胚胎中的表达。2)生成并表征galnac -4-硫转移酶缺陷小鼠。3)明确galnac -4-磺基转移酶的结构与功能关系。4)克隆蛋白特异性β 1,4- galnac -转移酶并表征其肽识别决定因素，利用一种新的分泌嵌合蛋白直接筛选转染细胞后的β 1,4- galnac -转移酶的表达。在整个脊椎动物进化过程中，糖蛋白激素上这些硫酸化结构的保存，以及识别它们的受体，证明了它们的重要性。我们将结合遗传、生化和结构方法来研究这些寡糖的生物学作用，这将促进我们对它们在下丘脑-垂体-性腺轴调节雌激素产生中的作用的理解。由于LH和TSH清除的改变，这些结构合成的改变预计会导致激素失衡。由于大脑和淋巴结树突状细胞中以GaINAc-4-S04终止的糖蛋白结构的高度调控表达，这种改变也可能导致大脑发育和对抗原的免疫反应异常。我们的研究还将揭示这些硫酸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.