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

• 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
• 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

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（由适用提供）：粘蛋白型甘氨酸是在还原末端通过N-乙酰基半乳糖胺连接到肽中的Ser或Thr的粘合聚糖。它们在分泌的粘蛋白和膜结合的糖蛋白中，包括一些粘蛋白。膜相关的粘蛋白型聚糖的一个主要功能是指导循环白细胞迁移到损伤部位和淋巴组织的位点，通过含有含有非还原末端的siAllyl Lewis X的相互作用。粘蛋白型聚糖在分泌的粘蛋白中的主要功能是通过保留水来保护粘液酶组织，并捕获和清除空气传播和注入的病原体。两种类型的聚糖的功能在很大程度上由6GlCNAC分支结构在很大程度上控制；对于与膜相关的粘蛋白型甘氨酸，它是核心2，对于在分泌的粘蛋白中发现的粘蛋白型聚糖，它是核心2，核心4和血型I抗原。 CORE 2可以通过Core 2 N-乙酰葡萄糖转移（C2GNT）-L，-M和-T同工酶合成CORE 2，由C2GNT-M和IGNT合成。因此，这些分支酶表达的改变可能会对生物学上重要的糖基质的产生产生重大影响，从而对粘蛋白的功能产生重大影响。目前尚不清楚为什么膜结合的突变仅包含核心2，而分泌的粘蛋白包含所有三个分支结构。文献信息表明，每种粘蛋白独有的非倾角重复肽序列以及高尔基堆栈中的微环境，其中分支酶驻留是关键的确定剂。该应用的目的是检验以下假设：秘密和膜结合的粘蛋白的粘蛋白聚糖分支结构的合成受C2GNT-M和C2GNT-L的不同亚高尔基体的控制。 The specific aims of this application are to demonstrate that: (1) overexpression of C2GnT-M would not affect the levels of core 2 structures in MUC1 and overexpression of C2GnT-L would not affect the levels of core 2 structures 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）通过免疫元电子显微镜将C2GNT-L和C2GNT-M定位在单独的亚golgi室。表达C2GNT-L，C2GNT-M，MUC1和MUC5AC的H292-MUC1细胞将用作细胞模型。 HPLC和MALDI-TOF-MS将分析从MUC1和MUC5AC释放的粘蛋白聚糖。通过用C2GNT-L代替其N末端区域，将生成要针对C2GNT-L位置的C2GNT-M。将类似制备要针对核心1合酶位置的核心3合酶。这一假设的证明将促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，这可以帮助制定策略来设计理论，以治疗与变化的肌动蛋白糖基化相关的肺部疾病患者。公共卫生相关性：提出的工作可以提高我们对控制粘蛋白碳氢化物（粘蛋白功能的主要决定剂）的关键因素的基本理解。获得的知识可以帮助设计策略来开发治疗剂，以治疗患有粘液性肺部疾病和癌症的患者。