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

描述（由申请人提供）：粘蛋白型聚糖是通过n-乙酰半乳糖胺在肽的还原端连接到丝氨酸或苏氨酸的共轭聚糖。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。膜相关粘蛋白型聚糖的一个主要功能是通过位于非还原末端的含有唾液Lewis x的聚糖与选择素的相互作用，引导循环白细胞迁移到损伤部位和淋巴组织。黏液型聚糖在分泌黏液中的主要功能是通过保留水分、捕获和清除空气传播和注射的病原体来保护黏液分泌组织。两类聚糖的功能在很大程度上受glcnac分支结构的控制；对于膜相关的粘蛋白型聚糖，它是核心2，对于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I型抗原。core2可由core2 n -乙酰氨基葡萄糖转移酶(C2GnT)- l、-M和- t同工酶合成，core4仅由C2GnT-M合成，而core4抗原可由C2GnT-M和IGnT合成。因此，这些分支酶表达的改变可以对生物学上重要的糖基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粘蛋白只包含核心2，而分泌粘蛋白包含所有三个分支结构。文献资料表明，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堆微环境是关键决定因素。本应用的目的是验证分泌和膜结合粘蛋白中粘蛋白聚糖分支结构的合成是由C2GnT-M和C2GnT-L的不同亚高尔基定位控制的假设。本应用程序的具体目的是证明：(1)过表达C2GnT-M不会影响MUC1中核心2结构的水平，过表达C2GnT-L不会影响MUC5AC中核心2结构的水平；(2)将C2GnT-M和core 3合成酶靶向到C2GnT-L和core 1合成酶的亚高尔基位点，分别产生含有核心4的MUC1；(3)免疫金电镜观察C2GnT-L和C2GnT-M分别定位于不同的亚高尔基区室。将表达C2GnT-L、C2GnT-M、MUC1和MUC5AC的H292-MUC1细胞作为细胞模型。MUC1和MUC5AC释放的粘蛋白聚糖采用HPLC和Maldi-Tof-Ms进行分析。将C2GnT-M的n端区域替换为C2GnT-L的n端区域，生成C2GnT-M定位C2GnT-L。将core3合酶定位到core1合酶位置，同样制备。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如何被控制的理解，这可以帮助制定治疗策略，以治疗与粘蛋白糖基化改变相关的肺部疾病患者。公共卫生相关性：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来开发治疗药物，以治疗粘液高分泌性肺病和癌症患者。

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.