Mucin Site Specific O-glycosylation

描述（由申请人提供）：含有高度O-糖基化粘蛋白结构域的糖蛋白具有重要的生物学作用，包括保护上皮细胞表面、调节细胞-细胞相互作用以及调节炎症和免疫应答。它们在肿瘤的发生和转移中也起重要作用，是常见的肿瘤抗原。这些糖蛋白中的O-糖基化结构域是其适当生物功能所必需的，主要是因为O-聚糖赋予了其扩展的蛋白酶抗性结构。ppGalNAc转移酶的大家族（>20个成员）通过将1- GalNAc添加到肽核心的Ser或Thr残基来启动O-糖基化的第一步。随后的转移酶通过将糖依次添加到肽连接的GalNAc来延长聚糖链。最近，许多ppGalNAc T的突变与结肠癌患者相关。由于结肠粘蛋白（MUC 2）敲除小鼠容易发展结肠癌，我们提出突变的h-ppGalNAc Ts不能适当地糖基化h-MUC 2，导致具有改变的性质（或稳定性）的结肠粘蛋白，从而导致疾病倾向增加。在这项工作中，突变型和野生型转移酶的酶和生物学特性将被表征，以了解它们的生物学功能（即在MUC 2糖基化中的作用）和随后在疾病中的作用。预计这些研究将导致用于检测、预防和/或治疗结肠癌和其他疾病的潜在模式。在目的1中，将表征一系列野生型ppGalNAc T肽和糖肽底物特异性，所述野生型ppGalNAc T与人类疾病有关或具有显著感兴趣的生物化学或生物学性质。在目的2中，将进一步表征与结肠癌遗传相关的突变型ppGalNAc T’s的酶学、底物特异性（包括MUC 2）和一般细胞生物学（即亚细胞定位和分泌）。这个目标的目标是最终了解这些突变转移酶与MUC 2糖基化和结肠癌的关联。这项工作也将提供这类转移酶的第一个详细的生化结构-功能分析。在目的3中，研究扩展到表征转移酶的糖肽底物偏好，所述转移酶将随后的糖转移到肽连接的GalNAc，即（2-Gal（1-3）、2-GlcNAc（1-3）和1-NeuNAc（2-6）），从而启动0-聚糖生物合成的第一个延伸步骤。这些转移酶（即分别为核心1、核心3和ST 6 GalNAc I和II转移酶）的改变也与疾病和各种癌症相关。作为这项工作的结果，肽序列和局部环境对O-聚糖延伸的初始步骤的调制的作用的基本理解将被获得。这项工作将有助于阐明这些转移酶的生物学作用及其对疾病的影响。这些研究也将产生用于合理预测位点特异性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，以了解它们在这种疾病中的生物学功能和作用。预计这些研究将导致用于检测、预防和/或治疗结肠癌和其他疾病的潜在模式。

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.