导　　师： 张延

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 上海交通大学

摘　　要： 糖基化是细胞内重要的翻译后修饰过程,细胞内有超过50/%的蛋白质可被糖基化。在许多生命过程中,糖链发挥了复杂的功能,如调节蛋白质折叠与稳定、淋巴归巢、细胞黏连与迁移、宿主-病原体相互作用、免疫应答、生物体繁殖和胚胎形成等。 糖基化分为N-糖基化和O-糖基化两种,多肽: N-乙酰氨基半乳糖基转移酶/(ppGalNAc-T, EC 2.4.1.41/)是催化粘蛋白型O-糖基化修饰起始的糖基转移酶。ppGalNAc-T是一个酶家族,每个成员都有不同的组织表达分布和底物特异性。本文研究了该酶家族的一个新亚家族——Y亚家族基因的生化特性及功能。Y亚家族由ppGalNAc-T8、-T9、-T17和-T18组成,与其它ppGalNAc-T相比,它们在氨基酸序列水平上存在一些重要的突变。遗传进化分析显示,Y亚家族基因仅存在于脊椎动物中,有趣的是,它们均失去了体外GalNAc转移活性。对Y亚家族新成员ppGalNAc-T18的模拟结构分析表明UDP-GalNAc结合位点的突变可能是导致Y亚家族酶失去催化活性的原因。另外,本文证明ppGalNAc-T18定位于内质网而不是高尔基体。我们对Y亚家族酶的功能进行了探讨,虽然还没有得到最后的结论,但其成员特殊的表达定位暗示了该亚家族在脊椎动物中发挥着重要的作用。 目前, ppGalNAc-T的底物研究是糖生物学领域的重要课题之一,它对揭示O-糖基化的功能具有重大意义。叠氮化糖基转移酶供体底物类似物标记是一种新的糖基化检测方法,叠氮基团可与炔基发生特异的Click反应,从而被标记炔基的荧光物质检测。该方法比常用的抗体或凝集素检测更加稳定且特异。本文结合叠氮化ppGalNAc-T供体底物类似物UDP-GalNAz检测手段和蛋白质芯片技术,利用可糖基化多肽芯片进行实验并优化实验条件,最终成功建立了基于蛋白质芯片的ppGalNAc-T底物高通量筛选方法,在16000个蛋白质中成功筛选出226个 Glycosylation is a significant post-translational modification in cells and more than 50/% of eukaryotic proteins are glycosylated. Glycans possess complex functions in a variety of biological conditions, such as intracellular protein folding and stability, lymphocyte trafficking, cell adhesion and migration, host-pathogen interaction, immune response, fertilization, and embryogenesis. Glycosylation can be classified into two common forms, N-glycosylation and O-glycosylation. The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase /(ppGalNAc-T, EC 2.4.1.41/) family and each member have unique expression pattern and substrate specificity. In this report, the function and the characteristic of a new subfamily gene of ppGalNAc-Ts, designated as Y subfamily, were researched. The Y subfamily consists of four members, they are ppGalNAc-T8, -T9, -T17, and -T18 which have significant mutations at the protein level compared with other ppGalNAc-Ts. Genetic analysis revealed that Y subfamily members only exist in vertebrate. Interestingly, all four Y subfamily members lack the classical in vitro GalNAc-transferase activity. Structural analysis by homology modeling of the defining member of Y subfamily, ppGalNAc-T18, suggested that mutations in the UDP-GalNAc binding pocket of Y subfamily may lead to the lost of their transferase activity. Furthermore, ppGalNAc-T18 localizes in ER rather than Golgi apparatus in lung carcinoma cells. We also study the biological function of Y subfamily, although the function has not been fully understood, their distinct expression indicates that they might have critical roles in O-glycosylation of vertebrate. Nowadays, the substrates research of ppGalNAc-T is a hot topic of Glycobiology research. It is significant to our understanding of the functions of O-glycosylation. Azido labeled substrate analog of glycosyltransferase is a new glycosylation detection method which is more specific and stable than trad

关 键 词： 糖基转移酶 多肽 乙酰氨基半乳糖转移酶 糖基化 粘蛋白 内质网定位 反应 蛋白质芯片 高通量

领　　域： [生物学]