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）是高等生物翻译后普遍存在的重要修饰。大多数通过分泌途径的蛋白质都被多种具有多种生物学功能的粘蛋白型o -聚糖修饰。因此，许多生物学过程和疾病状态都与o糖基化的正常或异常有关，包括冠状动脉疾病、肾脏功能的调节、器官发生、胚胎发育、多种癌症和生育。重要的是，单个o -聚糖启动和延长转移酶的缺失在果蝇和小鼠中分别具有发育致死性。目前还不清楚这些转移酶是如何选择它们的特定靶标的，以及它们的底物的哪些特征调节了它们的活性。这种在分子水平上的理解对于推断o -糖基化的生物学作用和预测o -糖基化位点是必要的。通过了解参与底物选择的所有因素，将为开发治疗异常o糖基化疾病（包括癌症）的新型和选择性策略开辟新的途径。此外，预测o -糖基化转移酶特异性位点的能力对于解释o -糖蛋白组学数据以及从全基因组关联研究（GWAS）中确定与疾病相关的糖基转移酶的靶标将是非常宝贵的。计划中的研究将集中于启动o糖基化的GalNAc-Ts大家族（T1-T20）和执行o糖基化第一步的核心转移酶（C1GALT1, B3GNT6， GCNT1和ST6GalNAc-1 & 2）。本研究旨在表征GalNAc-Ts独特的肽和糖肽底物特异性，并确定可能控制o糖基化的其他底物特征，如聚集电荷和预先糖基化。我们的主要工作假设是，o -聚糖的位点选择和特定延伸是由肽的性质调节的，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底物结合或建模到GalNAc-Ts和核心延长转移酶的晶体结构相关联来实现。进一步的研究将包括表征先前的苏氨酸和丝氨酸o糖基化的作用，酪氨酸残基的糖基化以及进一步完善我们基于网络的异构体特异性o糖基化预测工具ISOGlyP。最后，细胞序列工程将用于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特性、它们如何选择它们的靶点以及它们在疾病中的生物学作用和功能的最终机制的理解，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.