Initiation and Regulation of Mucin-Type O-Glycosylation

粘蛋白型蛋白O-糖基化(以下简称O-糖基化)是高等生物普遍存在的一种重要的翻译后修饰。大多数通过分泌途径的蛋白质都被一系列具有不同生物学功能的粘蛋白O-糖链所修饰。因此，许多生物学过程和疾病状态与正常或异常的O-糖基化有关，包括冠状动脉疾病、肾功能调节、器官发生、胚胎发育、多发性癌症和生育。重要的是，单个O-葡聚糖起始转移酶和延伸转移酶的缺失分别对苍蝇和小鼠的发育是致命的。目前，人们还不清楚这些转移酶是如何选择它们的特定靶标的，以及它们底物的哪些特性调节它们的活性。在分子水平上，这样的理解对于推断O-糖基化的生物学作用和预测O-糖基化的位置是必要的。通过了解底物选择中涉及的所有因素，将为开发新的选择性策略来治疗包括癌症在内的异常O-糖基化疾病开辟新的途径。此外，预测O-糖基化转移酶特定位点的能力对于解释O-糖蛋白组学数据和从全基因组关联研究(GWAS)中识别与疾病相关的糖基转移酶靶标将是非常有价值的。计划中的研究将集中在启动O-糖基化的GalNAc-ts(T1-T20)大家族和执行O-糖链延伸第一步(S)的核心转移酶(C1GALT1、B3GNT6、GCNT1和ST6GalNAc-1&2)上。本研究旨在鉴定GalNAc-ts独特的多肽和糖肽底物的特异性，并确定可能控制O-糖基化的其他底物特征，如簇状电荷和先前的糖基化。我们的主要工作假设是O-葡聚糖位点的选择和比伸长受多肽的性质调节，其主要成分是受体位点两侧残基的电荷分布。这项工作将提供对GalNAc-T底物选择的前所未有的理解，通过将我们的特异性和动力学数据与结合或模拟到GalNAc-T和核心延伸转移酶的底物的晶体结构相关联来实现。其他研究将包括表征先前苏氨酸与丝氨酸O-糖基化的作用，酪氨酸残基的糖基化，以及我们基于网络的异构体特异性O-糖基化预测工具ISOGlyP的进一步完善。最后，细胞中的序列工程将被用来证实我们的体外预测。总之，这些基础研究将极大地促进我们对这些转移酶的性质以及它们如何选择靶点以及最终它们在疾病中的生物学作用和功能的机制的理解，最终目标是开发出治疗异常O-糖基化疾病的有效疗法。

Mucin-type protein O-glycosylation (henceforth called O-glycosylation) is a ubiquitous and essential post translational modification of higher organisms. Most proteins passing through the secretory pathway are decorated with a wide range of mucin-type O-glycans which serve diverse biological functions. Hence, many biological processes and disease states are linked to normal or abnormal O-glycosylation including coronary artery disease, the regulation of kidney function, organogenisis, embryonic development, multiple cancers and fertility. Importantly, the loss of single O-glycan initiating and elongating transferases is developmentally lethal in the fly and mouse, respectively. Presently it is not well understood how these transferases chose their specific targets and what features of their substrates modulate their activities. Such an understanding, at the molecular level, is necessary for deducing the biological roles of O-glycosylation and for predicting sites of O-glycosylation. By understanding all of the factors involved in substrate selection new avenues will open for the development of novel and selective strategies to treat diseases of aberrant O-glycosylation including cancers. Furthermore, the ability to predict transferase specific sites of O-glycosylation will be invaluable for the interpretation of O-glycoproteomics data and for identifying the targets of glycosyltransferases linked to disease from genome wide association studies (GWAS). The planned research will focus on the large family of GalNAc-Ts (T1-T20) that initiate O-glycosylation and the core transferases (C1GALT1, B3GNT6, GCNT1 and ST6GalNAc-1 & 2) that perform the first step(s) of O-glycan elongation. This research aims to characterize the unique peptide and glycopeptide substrate specificities of the GalNAc-Ts as well as to identify additional substrate features such as clustered charges and prior glycosylation that may control O-glycosylation. Our major working hypothesis is that O-glycan site selection and specific elongation are modulated by the properties of the peptide with a major component being the charge distribution of residues flanking the acceptor site. This work will provide an unprecedented understanding of GalNAc-T substrate selection, achieved by correlating our specificity and kinetics data with the crystal structures of substrates bound or modeled onto the GalNAc-Ts and the core elongating transferases. Additional studies will involve characterizing the role of prior Thr versus Ser O-glycosylation, the glycosylation of Tyr residues and the further refinement of our web based isoform specific O-glycosylation prediction tool ISOGlyP. Finally, sequon engineering in cells will be utilized to confirm our in vitro predictions. Together, these basic studies will greatly advance our understanding of the properties of these transferases and how they chose their targets and ultimately the mechanisms of their biological role and function in disease with an eventual goal to develop useful therapeutics for the treatment of diseases of aberrant O-glycosylation.