Control of Mucin Glycan Branching in Membrane-bound and Secreted Mucins

膜结合和分泌粘蛋白中粘蛋白聚糖分支的控制

基本信息

批准号：
7924753
负责人：
PI-WAN CHENG
金额：
$ 19.91万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7924753
关键词：
Acetylgalactosamine Acetylglucosamine Affect Binding Biological Process Blood typing procedure Breathing Carbohydrates Cell model Cells Data Disease Electron Microscopy Environment Enzymes Epithelial Cells Epithelium Glycoconjugates Glycoproteins Goals Golgi Apparatus Grant High Pressure Liquid Chromatography I-antigen Injury Isoenzymes Knowledge Leukocyte Trafficking Leukocytes Link Literature Location Lung diseases Lymphoid Tissue MUC5AC gene Malignant Neoplasms Membrane Modeling Mucin-1 Staining Method Mucin-2 Staining Method Mucins Mucous body substance N-terminal Patients Peptides Polysaccharides Production Proteins Role Selectins Site Structure Tandem Repeat Sequences Testing Therapeutic Therapeutic Agents Thymus Gland Tissues Transferase Water Work base blood group design glycosylation migration overexpression pathogen protein aminoacid sequence public health relevance sialyl Lewis x trafficking

项目摘要

DESCRIPTION (provided by applicant): Mucin-type glycans are conjugated glycans linked through N-acetylgalactosamine at the reducing end to ser or thr in the peptide. They are found primarily in secreted mucins and membrane-bound glycoproteins, including some mucins. One major function of membrane-associated mucin-type glycans is to guide the migration of circulating leukocytes to site of injury and lymphoid tissues through interaction of sialyl Lewis x- containing glycans located at the non-reducing termini with selectins. The major function of mucin-type glycans in secreted mucins is to protect mucus-secetroy tissues by retention of water, and trapping and clearance of airborne and injected pathogens. Functions of both types of glycans are controlled to a large extent by ¿6GlcNAc branch structures; for membrane-associated mucin-type glycans, it is core 2 and for mucin-type glycans found in secreted mucins, it is core 2, core 4, and blood group I antigen. Core 2 can be synthesized by core 2 N-acetylglucosaminyl transferase (C2GnT)-L, -M and -T isozymes, core 4 by C2GnT- M only, and I antigen by C2GnT-M and IGnT. Therefore, alteration of the expression of these branching enzymes can have a significant impact on the production of biologically important glycotopes and thus the functions of mucins. It is not clear why membrane-bound mucins contain only core 2 while secreted mucins contain all three branch structures. Literature information suggests that non-tandem repeat peptide sequence unique to each mucin and the microenvironment in the Golgi stacks where the branching enzymes reside are the key determinants. The goal of this application is to test the hypothesis that synthesis of mucin glycan branch structures in secreted and membrane-bound mucins is controlled by different sub-Golgi localization of C2GnT-M and C2GnT-L. The specific aims of this application are to demonstrate that: (1) overexpression of C2GnT-M would not affect the levels of core 2 structure in MUC1 and overexpression of C2GnT-L would not affect the levels of core 2 structure in MUC5AC, (2) targeting C2GnT-M and core 3 synthase to the sub-Golgi locations of C2GnT-L and core 1 synthase, respectively would generate MUC1 containing core 4, and (3) C2GnT-L and C2GnT-M are localized at separate sub- Golgi compartments by immunogold electron microscopy. H292-MUC1 cells which express C2GnT-L, C2GnT-M, MUC1, and MUC5AC will be used as the cell model. Mucin glycans released from MUC1 and MUC5AC will be analyzed by HPLC and Maldi-Tof-Ms. C2GnT-M to be targeted to C2GnT-L location will be generated by replacing its N-terminal region with that of C2GnT-L. Core 3 synthase to be targeted to core 1 synthase location will be similarly prepared. Proof of this hypothesis would advance our understanding of how synthesis of different mucin glycan branch structures is controlled, which can help develop strategies to design therapeutics to treat patients with lung diseases associated with altered mucin glycosylation. PUBLIC HEALTH RELEVANCE: The work proposed could advance our fundamental understanding of the key factors that control the synthesis of mucin carbohydrates, the main determinants of mucin functions. The knowledge gained could help design strategy to develop therapeutic agents to treat patients with mucus hypersecretory lung diseases and cancer.

描述（由申请人提供）：粘蛋白型聚糖是通过 N-乙酰半乳糖胺的还原端与肽中的丝氨酸或苏氨酸连接的缀合聚糖，它们主要存在于分泌性粘蛋白和膜结合糖蛋白中，包括一些主要的粘蛋白。膜相关粘蛋白型聚糖的功能是通过含有唾液酸Lewis x的相互作用引导循环白细胞迁移至损伤部位和淋巴组织位于非还原末端的聚糖在分泌性粘蛋白中的主要功能是通过保留水分以及捕获和清除两种类型聚糖的功能来保护粘液分泌组织。很大程度上是由 ¿ 6GlcNAc分支结构；对于膜相关粘蛋白型聚糖，它是核心2，对于分泌性粘蛋白中发现的粘蛋白型聚糖，它是核心2、核心4，并且核心2可以合成血型I抗原。 N-乙酰氨基葡萄糖转移酶 (C2GnT)-L、-M 和 -T 同工酶，核心 4 仅由 C2GnT-M 提供，I 抗原由 C2GnT-M 提供因此，这些分支酶表达的改变会对生物学上重要的糖表位的产生产生显着影响，从而对粘蛋白的功能产生显着影响。目前尚不清楚为什么膜结合粘蛋白在分泌时仅含有核心 2。粘蛋白含有所有三种分支结构。文献信息表明，每种粘蛋白独特的非串联重复肽序列和分支酶所在的高尔基体微环境是关键。本申请的目的是测试分泌型和膜结合型粘蛋白中粘蛋白聚糖分支结构的合成是由 C2GnT-M 和 C2GnT-L 的不同亚高尔基体定位控制的假设。本申请的具体目的是。证明：（1）C2GnT-M的过表达不会影响MUC1中核心2结构的水平，并且C2GnT-L的过表达不会影响核心2的水平MUC5AC 中的结构，(2) 将 C2GnT-M 和核心 3 合酶分别靶向 C2GnT-L 和核心 1 合酶的高尔基体亚位点，将生成含有核心 4 的 MUC1，(3) C2GnT-L 和 C2GnT-M 是通过免疫金电子显微镜定位于表达 C2GnT-L 的 H292-MUC1 细胞， C2GnT-M、MUC1 和 MUC5AC 将用作细胞模型，通过 HPLC 分析从 MUC1 和 MUC5AC 释放的粘蛋白聚糖，并通过 Maldi-Tof-Ms 生成靶向 C2GnT-L 的位置。将其 N 末端区域替换为核心 3 合酶的 N 末端区域，以靶向核心 1 合酶位置。这一假设将增进我们对如何控制不同粘蛋白聚糖分支结构的合成的理解，这有助于制定策略来设计治疗与粘蛋白糖基化改变相关的肺部疾病的患者。公共卫生相关性：所提出的工作可以推进我们的基础研究。了解控制粘蛋白碳水化合物合成的关键因素，即粘蛋白功能的主要决定因素，所获得的知识可以帮助设计开发治疗粘液分泌过多性肺病和癌症患者的治疗药物。