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被广泛的粘蛋白型O-聚糖修饰，其具有多种生物学功能。因此，许多生物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、多种癌症和生育。重要的是，单个O-聚糖起始和延伸转移酶的丧失分别在果蝇和小鼠中是发育致死的。目前还不清楚这些转移酶如何选择其特异性靶点以及其底物的哪些特征调节其活性。这种理解，在分子水平上，是必要的推导O-糖基化的生物学作用和预测的O-糖基化的网站。通过了解底物选择中涉及的所有因素，将为开发治疗异常O-糖基化疾病（包括癌症）的新的选择性策略开辟新的途径。此外，预测O-糖基化的转移酶特异性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）中鉴定与疾病相关的糖基转移酶的靶标将是非常宝贵的。计划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-聚糖延伸的第一步的核心转移酶（C1 GALT 1，B3 GNT 6，GCNT 1和ST 6 GalNAc-1 & 2）。本研究旨在表征GalNAc-Ts的独特肽和糖肽底物特异性，以及鉴定其他底物特征，如簇电荷和可能控制O-糖基化的先前糖基化。我们的主要工作假设是O-聚糖位点选择和特异性延伸由肽的性质调节，其中主要组分是受体位点侧翼残基的电荷分布。这项工作将提供一个前所未有的理解GalNAc-T底物的选择，实现了相关的特异性和动力学数据与底物的晶体结构绑定或建模的GalNAc-Ts和核心延伸转移酶。其他研究将涉及表征先前Thr相对于Ser O-糖基化的作用、Tyr残基的糖基化以及我们基于网络的亚型特异性O-糖基化预测工具ISOGlyP的进一步改进。最后，将利用细胞中的序列子工程来证实我们的体外预测。总之，这些基础研究将大大推进我们对这些转移酶的性质的理解，以及它们如何选择它们的靶点，并最终了解它们在疾病中的生物学作用和功能的机制，最终目标是开发用于治疗异常O-糖基化疾病的有用疗法。

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.