Initiation and Regulation of Mucin-Type O-Glycosylation

粘蛋白型 O-糖基化的启动和调节

基本信息

批准号：
10259867
负责人：
THOMAS A GERKEN
金额：
$ 33.81万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10259867
关键词：
Acetylgalactosamine Base Sequence Biochemical Biological Biological Process Cardiovascular Diseases Cardiovascular system Cell Communication Cell surface Cells Charge Colon Complex Coronary Arteriosclerosis Crystallization Data Defect Development Disease Drug Industry Embryonic Development Enzymes Family Fertility Funding Future Glycopeptides Glycoproteins Goals Hormonal Host Defense Human Immune response In Vitro Individual Inflammation Inflammatory Kinetics Knowledge Link Malignant Neoplasms Membrane Metabolic Methods Modeling Modification Molecular Mucins Mus Online Systems Organism Pathway interactions Pattern Peptides Play Polypeptide N-acetylgalactosaminyltransferase Polysaccharides Post-Translational Protein Processing Prevalence Process Property Protein Glycosylation Protein Isoforms Proteins Regulation Renal function Research Role Series Site Specificity Structure Substrate Specificity Testing Therapeutic Therapeutic Uses Transferase Tumor Markers Work base fly genome wide association study glycoproteomics glycosylation glycosyltransferase in vivo man neoplastic novel polypeptide protein aminoacid sequence protein degradation receptor sialylation sugar tool

项目摘要

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.

粘蛋白型蛋白O-糖基化（以下称为O-糖基化）是高等生物普遍存在且必需的翻译后修饰。大多数通过分泌途径的蛋白质都被多种粘蛋白型 O-聚糖修饰，这些粘蛋白型 O-聚糖具有多种生物学功能。因此，许多生物过程和疾病状态都与正常或异常的O-糖基化有关，包括冠状动脉疾病、肾功能的调节、器官发生、胚胎发育、多种癌症和生育能力。重要的是，单个 O-聚糖起始和延伸转移酶的损失分别对果蝇和小鼠的发育是致命的。目前尚不清楚这些转移酶如何选择其特定靶点以及其底物的哪些特征调节其活性。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）的新型选择性策略开辟新途径。此外，预测 O-糖基化转移酶特定位点的能力对于解释 O-糖蛋白组学数据以及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具有非常重要的价值。计划的研究将重点关注启动 O-糖基化的 GalNAc-Ts (T1-T20) 大家族以及执行 O-糖基化第一步的核心转移酶（C1GALT1、B3GNT6、GCNT1 和 ST6GalNAc-1 & 2）。 -聚糖伸长。本研究旨在表征 GalNAc-T 独特的肽和糖肽底物特异性，并确定其他底物特征，例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伸长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。这项工作将通过将我们的特异性和动力学数据与结合或建模到 GalNAc-T 和核心延伸转移酶上的底物的晶体结构相关联来实现对 GalNAc-T 底物选择的前所未有的理解。其他研究将涉及表征先前 Thr 与 Ser O-糖基化的作用、Tyr 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，细胞中的序列子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以及它们如何选择靶标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，最终目标是开发用于治疗异常 O 疾病的有用疗法。 -糖基化。