Initiation and Regulation of Mucin-Type O-Glycosylation

粘蛋白型 O-糖基化的启动和调节

• 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
• 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

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（此后称为O-糖基化）是一种无处不在的较高生物体转化后修饰。大多数穿过分泌途径的蛋白质都装饰有多种粘蛋白型O-聚糖，这些蛋白质具有多种生物学功能。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，肾功能的调节，有机基因，胚胎发育，多种癌症和生育能力。重要的是，单个O-聚糖启动和拉长转移酶的丧失在苍蝇和小鼠中分别是致命的。目前，尚不清楚这些转移酶如何选择其特定目标以及其底物的哪些特征调节其活动。在分子水平上，这种理解对于推断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，新途径将开辟新的和选择性策略，以治疗包括癌症在内的异常O-糖基化疾病。此外，预测O-糖基化转移酶特定位点的能力对于解释O-糖蛋白质组学数据以及鉴定与基因组广泛关联研究（GWAS）相关的糖基转移酶的靶标非常宝贵。计划的研究将集中于启动O-糖基化和核心转移酶（C1GALT1，B3GNT6，GCNT1和ST6GALNAC-1＆2）的大型GalNAC-TS（T1-T20），从而执行O-GlyCan延长的第一步。这项研究旨在表征GALNAC-TS的独特肽和糖肽底物特异性，并鉴定可能控制O-糖基化的其他底物特征，例如聚集电荷和先前的糖基化。我们的主要工作假设是，O-Glycan位点的选择和特定的伸长受肽的特性调节，主要组成部分是受体位点侧面残基的电荷分布。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建模到GalNAC-TS和核心延长转移酶结合或建模的底物的晶体结构相关联。其他研究将涉及表征先前的THR与ser o-糖基化的作用，Tyr残基的糖基化以及我们基于Web的同工型特异性O-糖基化预测工具Isoglyp的进一步细化。最后，将利用细胞中的序列工程来确认我们的体外预测。总之，这些基础研究将大大提高我们对这些转移酶特性的理解，以及它们如何选择其生物学作用和功能在疾病中的机制，最终目标是开发有用的治疗疗法，以治疗异常的O-糖基化疾病。