Mucin Site Specific O-glycosylation

描述(申请人提供)：含有大量O-糖基化粘蛋白结构域的糖蛋白发挥着重要的生物学作用，包括保护上皮细胞表面，调节细胞间的相互作用，以及调节炎症和免疫反应。它们也在肿瘤的发生和转移中发挥重要作用，是常见的肿瘤抗原。这些糖蛋白中的O-糖基化结构域是其正常生物学功能所必需的，主要是因为它们具有由O-糖链赋予的扩展的抗蛋白酶结构。一个ppGalNAc转移酶大家族(&gt；20个成员)通过在多肽核心的丝氨酸或苏氨酸残基上添加1-GalNAc来启动O-糖基化的第一步。随后的转移酶通过在多肽连接的GalNAc上依次添加糖来延长糖链。最近，一些ppGalNAc-ts的突变与结肠癌患者有关。由于结肠粘蛋白(MUC2)基因敲除的小鼠容易患结肠癌，我们认为突变的h-ppGalNAc ts不能正确地糖基化h-MUC2，导致结肠粘蛋白的性质(或稳定性)改变，导致疾病倾向增加。在这项工作中，将对突变型和野生型转移酶的酶和生物学特性进行表征，以了解它们的生物学功能(即在MUC2糖基化中的作用)以及随后在疾病中的作用。预计这些研究将导致检测、预防和/或治疗结肠癌和其他疾病的潜在方法。在AIM 1ppGalNAc T肽和糖肽底物的特异性将表征的一系列野生型ppGalNAc T已牵涉到人类疾病或具有重要的生化或生物学特性。在目标2中，突变的ppGalNAc T‘s在基因上与结肠癌有关，将进一步从其酶学、底物特异性(包括MUC2)和一般细胞生物学(即亚细胞定位和分泌)方面进行表征。这一目标的目的将是最终了解这些突变转移酶与MUC2糖基化和结肠癌的关联。这项工作还将首次对这类转移酶进行详细的生化结构和功能分析。在AIM中，3项研究被扩展以表征转移酶的糖肽底物偏好，这些转移酶将随后的糖转移到与GalNAc相连的多肽上，即(2-Gal(1-3)，2-GlcNAc(1-3)和1-NeuNAc(2-6))，启动O-糖聚糖生物合成的第一个延伸步骤。这些转移酶(即分别是核心1、核心3和ST6GalNAc I和II转移酶)的改变也与疾病和各种癌症有关。作为这项工作的结果，将获得对肽序列和局部环境在调节O-葡聚糖延伸的初始步骤中的作用的基本了解。这项工作将有助于阐明这些转移酶的生物学作用(S)及其对疾病的影响。这些研究还将产生合理预测特定部位O-糖基化的工具。公共卫生相关性：最近在结肠癌患者中发现了一系列糖基转移酶(蛋白质糖基化酶)的突变。在这项工作中，我们将研究一系列突变型和野生型转移酶的酶学和生物学特性，以了解它们在本病中的生物学功能和作用。预计这些研究将导致检测、预防和/或治疗结肠癌和其他疾病的潜在方法。

DESCRIPTION (provided by applicant): Glycoproteins containing heavily O-glycosylated mucin domains play important biological roles including protecting epithelial cell surfaces, modulating cell-cell interactions and regulating the inflammatory and immune responses. They also play important roles in tumorigenesis and metastasis and are common tumor antigens. The O-glycosylated domains in these glycoproteins are required for their proper biological function, principally because of their extended protease resistant structures imparted by the O-glycans. A large family (>20 members) of ppGalNAc transferases initiates the first step of O-glycosylation by adding 1- GalNAc to Ser or Thr residues of the peptide core. Subsequent transferases elongate the glycan chain by sequentially adding sugars to peptide linked GalNAc. Recently mutations in a number of ppGalNAc Ts have been associated with patients with colon cancer. Since the colonic mucin (MUC2) knock-out mouse readily develops colon cancer, we propose that the mutated h-ppGalNAc Ts fail to properly glycosylate h-MUC2, resulting in a colonic mucin with altered properties (or stability) leading to an increased propensity for disease. In this work the enzymatic and biological properties of the mutant and wild type transferases will be characterized in order to understand their biological functions (i.e. role in MUC2 glycosylation) and subsequent role in disease. It is anticipated that these studies will lead to potential modalities for the detection, prevention, and/or treatment of colon cancer and other diseases. In Aim 1 ppGalNAc T peptide and glycopeptide substrate specificities will be characterized for a series of wild type ppGalNAc Ts that have been implicated in human disease or have significantly interesting biochemical or biological properties. In Aim 2 the mutant ppGalNAc T's, genetically linked to colon cancer, will be further characterized with respect to their enzymology, substrate specificities (including MUC2) and general cell biology (i.e. subcellular location and secretion). The goals of this aim will be to ultimately understand the association of these mutant transferases with MUC2 glycosylation and colon cancer. This work will also provide the first detailed biochemical structure-function analysis of this class of transferases. In Aim 3 studies are extended to characterize the glycopeptide substrate preferences of transferases that transfer subsequent sugars to the peptide linked GalNAc i.e. (2-Gal (1-3), 2-GlcNAc (1-3) & 1-NeuNAc (2-6)), initializing the first elongation steps of O-glycan biosynthesis. Alterations in these transferases (i.e. the Core 1, Core 3, and ST6GalNAc I & II transferases, respectively) also have associations with disease and various cancers. As a result of this work a basic understanding of the role of peptide sequence and local environment on the modulation of the initial steps of O-glycan elongation will be obtained. This work will contribute to the elucidation of the biological role(s) of these transferases and their impact on disease. Tools for the rational prediction of site specific O-glycosylation will also result from these studies. PUBLIC HEALTH RELEVANCE: Mutations in a series of glycosyltransferases (protein glycosylating enzymes) have recently been found in patients with colon cancer. In this work we will study the enzymatic and biological properties of a series of mutant and wild type transferases to understand their biological functions and roles in this disease. It is anticipated that these studies will lead to potential modalities for the detection, prevention, and/or treatment of colon cancer and other diseases.