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

性状（由申请方提供）：粘蛋白型聚糖是通过N-乙酰半乳糖胺在还原端与肽中的ser或thr连接的共轭聚糖。它们主要存在于分泌性粘蛋白和膜结合糖蛋白中，包括一些粘蛋白。膜相关粘蛋白型聚糖的一个主要功能是通过位于非还原末端的含唾液酸刘易斯x的聚糖与选择素的相互作用，引导循环白细胞迁移至损伤部位和淋巴组织。分泌型粘蛋白中的粘蛋白型聚糖的主要功能是通过保持水分以及捕获和清除空气传播和注射的病原体来保护粘液分泌组织。这两种类型的聚糖的功能在很大程度上受分支结构的控制;对于膜相关粘蛋白型聚糖，它是核心2，对于分泌型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。核心2可由核心2 N-乙酰葡糖胺基转移酶（C2 GnT）-L、-M和-T同工酶合成，核心4仅由C2 GnT-M合成，而I抗原由C2 GnT-M和IGnT合成。因此，这些分支酶的表达的改变可以对生物学上重要的糖表位的产生以及粘蛋白的功能具有显著影响。目前尚不清楚为什么膜结合粘蛋白只含有核心2，而分泌粘蛋白含有所有三个分支结构。文献信息表明，每个粘蛋白所特有的非串联重复肽序列和分支酶所在的高尔基体中的微环境是关键决定因素。本申请的目的是检验分泌型和膜结合型粘蛋白中粘蛋白聚糖分支结构的合成受C2 GnT-M和C2 GnT-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）C2 GnT-M的过表达不会影响MUC 1中核心2结构的水平，C2 GnT-L的过表达不会影响MUC 5AC中核心2结构的水平，（2）将C2 GnT-M和核心3合酶分别靶向C2 GnT-L和核心1合酶的亚高尔基体位置将产生含有核心4的MUC 1，（3）免疫金电镜显示C_2GnT-L和C_2GnT-M分别定位于高尔基体亚区室。将表达C2 GnT-L、C2 GnT-M、MUC 1和MUC 5AC的H292-MUC 1细胞用作细胞模型。将通过HPLC和Maldi-Tof-Ms.分析从MUC 1和MUC 5AC释放的粘蛋白聚糖。将通过用C2 GnT-L的N-末端区域替换其N-末端区域来产生靶向C2 GnT-L位置的C2 GnT-M。将类似地制备靶向核心1合酶位置的核心3合酶。这一假设的证明将促进我们对不同粘蛋白聚糖分支结构的合成是如何控制的理解，这可以帮助开发策略来设计治疗与改变的粘蛋白糖基化相关的肺部疾病的治疗方法。公共卫生相关性：提出的工作可以推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，开发治疗粘液分泌过多性肺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.