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

说明书(申请人提供)：粘蛋白类型的多糖是通过N-乙酰半乳糖胺在还原末端连接在多肽中服务或THR的结合多糖。它们主要存在于分泌的粘蛋白和膜结合的糖蛋白中，包括一些粘蛋白。膜相关粘蛋白糖链的一个主要功能是通过位于非还原末端的唾液酸化Lewis x糖链与选择素的相互作用，引导循环中的白细胞向损伤部位和淋巴组织迁移。分泌型粘蛋白中的粘蛋白多聚糖的主要功能是通过保持水分、捕获和清除空气传播的和注射的病原体来保护粘液组织。这两种类型的多糖的功能在很大程度上受6GlcNAc分支结构的控制；对于膜相关粘蛋白类型的多糖来说，它是核心2，对于在分泌的粘蛋白中发现的粘蛋白类型的多糖来说，它是核心2、核心4和血型I型抗原。核心2由核心2的N-乙酰氨基葡萄糖基转移酶(C2GnT)-L、-M和-T同工酶合成，核心4仅由C2GnT-M合成，核心1由C2GnT-M和IGnT合成。因此，这些分支酶的表达变化可以对生物上重要的糖蛋白的产生产生重大影响，从而影响粘蛋白的功能。目前尚不清楚为什么膜结合的粘蛋白只包含核心2，而分泌的粘蛋白包含所有三个分支结构。文献信息表明，每个粘蛋白特有的非串联重复序列和分支酶所在的高尔基体堆叠中的微环境是关键决定因素。这项应用的目的是检验一种假设，即分泌型和膜结合型粘蛋白中粘蛋白多糖分支结构的合成受C2GnT-M和C2GnT-L的不同亚高尔基定位控制。本申请的具体目的是证明：(1)过表达C2GnT-M不会影响MUC1中核心2结构的水平，C2GnT-L的过表达不会影响MUC5AC中核心2结构的水平，(2)将C2GnT-M和核心3合酶分别靶向C2GnT-L和核心1合酶的亚高尔基位置，会产生含有核心4的MUC1，以及(3)免疫组化电子显微镜下，C2GnT-L和C2GnT-M定位于单独的亚高尔基间隔。以表达C2GnT-L、C2GnT-M、MUC1、MUC5AC的H292-MUC1细胞为细胞模型。从MUC1和MUC5AC释放的粘蛋白多糖将用高效液相色谱和MALDI-TOF-MS进行分析。针对C2GnT-L位置的C2GnT-M将通过将其N-末端区域替换为C2GnT-L而产生。将类似地制备针对核心1合酶位置的核心3合酶。这一假说的证明将有助于我们理解不同粘蛋白多糖分支结构的合成是如何被控制的，这有助于开发策略来设计治疗与粘蛋白糖基化改变相关的肺部疾病的药物。与公共健康相关：提出的工作可以促进我们对控制粘蛋白碳水化合物合成的关键因素的基本理解，粘蛋白碳水化合物是粘蛋白功能的主要决定因素。所获得的知识可以帮助设计策略，开发治疗粘液过度分泌的肺部疾病和癌症患者的治疗药物。

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.