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: 8833545
• 负责人: THOMAS A GERKEN
• 金额: $ 30.67万
• 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 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8833545
• 关键词: Accounting; Address; Animal Model; Animals; Belief; Benign; Biological; Biological Process; Biology; C-terminal; Cardiovascular Diseases; Cardiovascular system; Cell Communication; Cell Culture Techniques; Cell Line; Cells; Complement; Data; Development; Disease; Embryonic Development; Enzymatic Biochemistry; Enzymes; Family; Fertility; Functional disorder; Glycopeptides; Goals; Health; Hormonal; Host Defense; Human; In Vitro; Individual; Inflammatory; Institution; Kinetics; Knock-out; Lead; Learning; Libraries; Link; Malignant Neoplasms; Membrane; Metabolic; Methods; Modification; Molecular; Mucins; Mus; Mutation; Online Systems; Pattern; Peptides; Play; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides; Prevalence; Process; Property; Protein Glycosylation; Protein Isoforms; Proteins; Regulation; Reporting; Reproduction; Role; Series; Site; Specificity; Structure; Substrate Specificity; Therapeutic; Tissues; Transferase; Work; animal tissue; base; design; fly; glycosylation; in vivo; inhibitor/antagonist; innovation; male; man; member; neoplastic; novel; novel strategies; polypeptide; ppGalNAc-T; predictive modeling; preference; public health relevance; sugar; therapeutic target; tool

 DESCRIPTION (provided by applicant): Most membrane and secreted proteins are decorated with mucin-type O-glycans which serve diverse biological roles. The mechanisms behind their biological roles and more specifically what governs O-glycan site selection and subsequent O-glycan elongation are largely unknown. The objective of this project is to elucidate the processes governing O-glycan site selection and O-glycan elongation at the enzyme and peptide substrate level in order to address the molecular mechanisms and biology of O-glycosylation. Mucin type O- glycosylation is vital to animal reproduction and development and increasingly linked to a wide range of rare to common disease states (such as hormonal/metabolic dysfunction, impaired host defense, inflammatory and cardiovascular diseases and even cancers) where it serves to modulate diverse biological functions including cell-cell interactions. Many disorders are linked to changes in expression or mutation of individual members of the large family (20 in man) of polypeptide-GalNAc transferases (ppGalNAc Ts) that initiate O-glycosylation by adding GalNAc to polypeptide Ser or Thr residues. O-glycosylation is absolutely required for embryonic development of the mouse and fly, relying on the Core 1 elongating transferase (T-synthase) in the mouse and several individual ppGalNAc T isoforms in the fly. How individual ppGalNAc T isoforms in this large family (or even T-synthase) can play such critical biological roles is unknown as the substrate specificity of these transferases have not been sufficiently characterized. That ppGalNAc T site selection is also modulated (positively or negatively) by prior glycosylation underscores its complexity and how much more needs to be learned of its specificity. The detailed characterization of ppGalNAc T isoform and elongating transferase specificity proposed in this project will lead to our understanding of the molecular mechanisms underlying the biological roles of O-glycosylation and will eventually lead to novel strategies to treat diseases of aberrant O- glycosylation. The AIMS of this project are to (1) expand the innovative use of a library of novel random peptide and glycopeptide substrates to fully characterize the specificity and basic enzymology of the transferases that initiate and elongate mucin type O-glycosylation, (2) to use these data to further develop sophisticated web based O-glycan predictive tools that include ppGalNAc T isoform and elongating transferase peptide and glycopeptide specificity, and (3) to develop a novel method for identifying in vivo isoform specific glycosylation targets using tissues from transferase knock-out animal models. These basic studies together with those of colleagues at other institutions will advance our understanding of the properties of these transferases, their targets (and resultant glycan structures) and ultimately the mechanisms of their biological role and function. These studies will significantly advance the field and will allw the development of novel specific inhibitors for potential use as targeted therapeutics.

 描述(申请人提供)：大多数膜和分泌的蛋白质都装饰有粘蛋白类型的O-多糖，具有不同的生物学作用。它们的生物学作用背后的机制，更具体地说，是什么控制了O-葡聚糖位点的选择和随后的O-葡聚糖延伸，在很大程度上是未知的。本项目的目的是在酶和肽的底物水平上阐明控制O-糖链位置选择和O-糖链延伸的过程，以解决O-糖基化的分子机制和生物学问题。粘蛋白O-糖基化对动物的繁殖和发育至关重要，并与一系列罕见到常见的疾病(如激素/代谢功能障碍、宿主防御功能受损、炎症性和心血管疾病，甚至癌症)密切相关，在这些疾病中，粘蛋白参与调节包括细胞-细胞相互作用在内的多种生物学功能。许多疾病与多肽-GalNAc转移酶(PpGalNAc Ts)大家族单个成员表达或突变的变化有关，这些多肽-GalNAc转移酶(PpGalNAc Ts)通过将GalNAc添加到多肽Ser或Thr残基来启动O-糖基化。O-糖基化是小鼠和苍蝇胚胎发育所必需的，依赖于小鼠的核心1延伸转移酶(T-合成酶)和苍蝇中的几个单独的ppGalNAc T亚型。在这个大家族中，单个ppGalNAc T亚型(甚至T-合成酶)如何发挥如此关键的生物学作用尚不清楚，因为这些转移酶的底物特异性还没有得到充分的表征。PpGalNAc T位点的选择也受到先前糖基化的调节(无论是积极的还是消极的)，这突显了它的复杂性，以及对其特异性需要更多的了解。本项目中提出的ppGalNAc T亚型和延伸转移酶特异性的详细表征将有助于我们理解O-糖基化的生物学作用的分子机制，并最终将导致治疗异常疾病的新策略 O-糖基化。该项目的目的是(1)扩大新的随机多肽和糖肽底物库的创新用途，以充分表征启动和延长粘蛋白O-糖基化的转移酶的特异性和基础酶学，(2)利用这些数据进一步开发复杂的基于网络的O-糖链预测工具，包括ppGalNAc T异构体和延长转移酶多肽和糖肽专一性，以及(3)开发一种利用组织鉴定体内异构体特异性糖基化靶标的新方法 来自转移酶基因敲除的动物模型。这些基础研究与其他机构的同事一起，将促进我们对这些转移酶的性质、它们的靶标(以及生成的多糖结构)以及最终它们的生物学作用和功能的机制的理解。这些研究将极大地推动该领域的发展，并将使新型特异性抑制剂的开发成为潜在的靶向治疗药物。