Glycoprotein Hormone Oligosaccharides

描述(由申请人提供)：这笔赠款的长期目标是了解一系列独特的碳水化合物结构的生物学作用，这些结构以S04-4-Ga1NAcBeta1，4GIcNAcBeta1，2Manpha序列终止。我们以前已经证明，所有脊椎动物的糖蛋白激素促甲状腺激素(TSH)和促黄体生成素(LH)上的N-连接碳水化合物都以这种独特的硫酸盐结构终止。此外，我们已经证明，这些硫酸盐结构控制着黄体生成素的循环半衰期，并且对于下丘脑-垂体-性腺轴调节雌激素的产生是必不可少的。我们现在已经克隆了两个GalNAc-4-磺基转移酶GaINAc-4-ST1和GaINAc-4-ST2，它们将硫酸盐转移到末端的β-1，4-连接的GaINAc。GaINAc-4-ST1在脑垂体中高度表达，并解释了在N-连接的低聚糖上存在GalNAc-4-S04的原因。由脑、肾、脾、淋巴结、卵巢、睾丸和子宫的细胞产生的终止于Beta1，4-连接的GaINAc-4-S04的额外糖蛋白表明，这些结构也将对其他生物过程至关重要。我们将：1)研究GainAc-4-磺基转移酶在成体和发育中胚胎中的表达。2)制备并鉴定GalNAc-4-磺基转移酶缺陷小鼠。3)确定GalNAc-4-磺基转移酶的结构：功能关系。4)克隆蛋白特异性的β1，4-GalNAc-转移酶并鉴定其多肽识别决定簇，利用一种新的分泌型嵌合蛋白直接筛选β1，4-GalNAc-转移酶的表达。在整个脊椎动物进化过程中，糖蛋白荷尔蒙上的这些硫酸盐化结构以及识别它们的受体的保存，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。由于黄体生成素和促甲状腺激素清除的改变，这些结构的合成改变预计会导致荷尔蒙失衡。这种改变也可能导致大脑发育异常和对抗原的免疫反应，这是因为在大脑和淋巴结的树突状细胞中，带有以GaINAc-4-S04终止的结构的糖蛋白的高度调控表达。我们的研究还将揭示这些硫化低聚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.