Initiation and regulation of mucin-type O-glycosylation

 描述（由申请方提供）：大多数膜蛋白和分泌蛋白均饰有粘蛋白型O-聚糖，其具有不同的生物学作用。其生物学作用背后的机制，更具体地说，是什么支配O-聚糖位点选择和随后的O-聚糖延伸在很大程度上是未知的。本项目的目的是阐明在酶和肽底物水平上O-聚糖位点选择和O-聚糖延伸的过程，以解决O-糖基化的分子机制和生物学。粘蛋白型0-糖基化对动物生殖和发育至关重要，并且越来越多地与广泛的罕见至常见疾病状态（如激素/代谢功能障碍、宿主防御受损、炎症和心血管疾病以及甚至癌症）相关，其中其用于调节包括细胞-细胞相互作用的多种生物功能。许多疾病与多肽-GalNAc转移酶（ppGalNAc Ts）的大家族（人类中有20个）的单个成员的表达或突变的变化有关，所述多肽-GalNAc转移酶通过将GalNAc添加到多肽Ser或Thr残基来启动O-糖基化。O-糖基化是小鼠和果蝇胚胎发育绝对需要的，依赖于小鼠中的核心1延伸转移酶（T-合酶）和果蝇中的几种单独的ppGalNAc T同种型。这个大家族中的单个ppGalNAc T同种型（或甚至T-合酶）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尚未得到充分表征。ppGalNAc T位点选择也受先前糖基化的调节（正或负），这强调了其复杂性以及需要了解其特异性的程度。该项目中提出的ppGalNAc T亚型和延伸转移酶特异性的详细表征将使我们理解O-糖基化的生物学作用的分子机制，并最终导致治疗异常糖基化疾病的新策略。 O-糖基化。该项目的目标是（1）扩展新型随机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型O-糖基化的转移酶的特异性和基本酶学，（2）使用这些数据进一步开发基于网络的复杂O-聚糖预测工具，包括ppGalNAc T同种型和延长转移酶肽和糖肽特异性，和（3）开发一种使用组织鉴定体内同种型特异性糖基化靶标的新方法 从转移酶敲除动物模型。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，其目标（和所得聚糖结构）以及最终其生物学作用和功能机制的理解。这些研究将显著推进该领域，并将允许开发新的特异性抑制剂作为靶向治疗的潜在用途。

 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.