Mucin Site Specific O-glycosylation

粘蛋白位点特异性 O-糖基化

• 批准号: 7816806
• 负责人: THOMAS A GERKEN
• 金额: $ 27.6万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-05 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7816806
• 关键词: Ablation; Acetylgalactosamine; Affect; Antigens; Binding Sites; Biochemical; Biological; Biological Process; Breast; CA-125 Antigen; CD8B1 gene; Calcinosis; Cancer Patient; Catalytic Domain; Cell Communication; Cell physiology; Cell surface; Cells; Cellular biology; Charge; Colitis; Collaborations; Colon; Colon Carcinoma; Colorectal Neoplasms; Common Neoplasm; Complement; Detection; Development; Disease; Drug or chemical Tissue Distribution; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Epithelial Cells; Family; Funding; Glycopeptides; Glycoproteins; Goals; Homologous Gene; Human; IgA1; Immune response; Immunoglobulin A; In Vitro; Individual; Inflammatory; Kinetics; Knockout Mice; Knowledge; Lead; Lectin; Link; Location; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Membrane Proteins; Methods; Modality; Modeling; Muc 2 protein; Mucin-1 Staining Method; Mucin-2 Staining Method; Mucins; Mus; Mutate; Mutation; N-acetylglucopyranosylamine; Neoplasm Metastasis; O-Glycans Biosynthesis Pathway; P-selectin ligand protein; PTPRC gene; Patients; Pattern; Peptide Hydrolases; Peptides; Phenotype; Play; Polysaccharides; Post-Translational Protein Processing; Predisposition; Prevention; Progress Reports; Property; Protein Isoforms; Proteins; Proteolysis; Research; Research Design; Resistance; Role; Sequence Analysis; Series; Site; Sorting - Cell Movement; Specificity; Structure; Substrate Specificity; Syndrome; Tandem Repeat Sequences; Tertiary Protein Structure; Transferase; Transforming Growth Factor beta Receptors; Tumor Antigens; United States National Institutes of Health; Work; fly; genome sequencing; glycosylation; glycosyltransferase; human disease; in vivo; interest; knowledge base; loss of function; malignant breast neoplasm; man; member; mutant; novel; novel strategies; polypeptide; ppGalNAc-T; preference; programs; protein aminoacid sequence; public health relevance; receptor; stem; sugar; tool; trafficking; tumor; tumorigenesis

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.

描述（由申请人提供）：含有大量O-糖基化粘蛋白结构域的糖蛋白起着重要的生物学作用，包括保护上皮细胞表面，调节细胞 - 细胞相互作用并调节炎症和免疫反应。它们在肿瘤发生和转移中也起着重要作用，并且是常见的肿瘤抗原。这些糖蛋白中的O-糖基化结构域是其适当的生物学功能所必需的，这主要是因为O-聚糖赋予其扩展的抗蛋白酶抗性结构。 PPGALNAC转移酶的大型家族（> 20个成员）通过将1- galnac添加到肽核的Ser或THR残基中，从而启动O-糖基化的第一步。随后的转移酶通过将糖顺序添加到肽连接的Galnac中，从而拉长了聚糖链。最近，许多PPGALNAC TS的突变与结肠癌患者有关。由于结肠粘蛋白（MUC2）敲除小鼠很容易发生结肠癌，因此我们建议突变的H- PPGALNAC TS无法正确地糖基化H-MUC2，从而导致结肠粘蛋白具有改变的特性（或稳定性），从而导致疾病的倾向增加。在这项工作中，将表征突变体和野生型转移酶的酶促和生物学特性，以了解其生物学功能（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结肠癌和其他疾病的检测，预防和/或治疗的潜在方式。在AIM 1中，将以一系列与人类疾病有关或具有显着有趣的生化或生物学特性的野生型PPGALNAC TS来表征一系列野生型PPGALNAC TS的特征。在AIM 2中，与结肠癌遗传有关的突变体PPGALNAC T将进一步特征在其酶学，底物特异性（包括MUC2）和一般细胞生物学（即亚细胞位置和分泌）方面。该目标的目标是最终了解这些突变转移酶与MUC2糖基化和结肠癌的关联。这项工作还将提供此类转移酶的第一个详细的生化结构 - 功能分析。在AIM 3中进行了扩展的研究，以表征转移糖的转移酶的糖肽底物偏好，这些酶将随后的糖转移到肽链接的GalNAC，即（2-GAL（1-3）（1-3），2-GLCNAC（1-3）和1-NEUNAC（2-6）），初始化o甘氨酸生物酸酯生物生物的第一个延长步骤。这些转移酶的改变（即核心1，核心3和ST6GALNAC I和II转移酶）也与疾病和各种癌症有关。由于这项工作，将获得对肽序列和局部环境在调制O-聚糖伸长初始步骤的作用的基本理解。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现场特定O-糖基化的合理预测的工具。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和野生型转移酶的酶促和生物学特性，以了解其生物学功能和在该疾病中的作用。预计这些研究将导致结肠癌和其他疾病的检测，预防和/或治疗的潜在方式。