Glycosyltransferase Golgi Retention Mechanism

描述（由申请人提供）： 慢性阻塞性肺疾病和肺癌是许多退伍军人的两大医疗问题。粘液分泌过多和转移是导致这些患者死亡的主要原因。这些疾病可由粘蛋白聚糖合成和结构的改变引起。在糖基转移酶的催化下，粘蛋白型聚糖从ser/thr以逐步的方式进行加工。其中，分支酶是独特的，因为它们合成26个N-乙酰氨基葡萄糖苷分支结构。这些酶包括核心2 N-乙酰葡糖胺基转移酶（C2 GnT）-1/L，C2 GnT-2/M和C2 GnT-3/T。C2 GnT-L合成膜糖蛋白中的核心2，C2 GnT-M合成分泌粘蛋白中的所有三个分支结构：核心2、核心4和I抗原，而C2 GnT-3的功能尚不清楚。C2 GnT-L的下调导致免疫功能缺陷，而过度表达导致癌症。C2 GnT-M的功能丧失导致结肠癌。C2 GnT-L和C2 GnT-M将是本次应用的重点。聚糖的合成不仅取决于糖基转移酶的数量，还取决于它们的高尔基体定位。糖基转移酶的亚高尔基体定位与其参与聚糖合成的步骤密切匹配。已知结肠癌中的糖基转移酶是错误靶向的，这导致短粘蛋白聚糖的形成。糖基转移酶的高尔基体定位信号主要存在于N端胞质尾区和跨膜区。然而，其机制尚不清楚。我们的初步研究表明，C2 GnT-L和C2 GnT-M在细胞内分离，即使两者都合成核心2并且预期共定位。此外，C2 GnT-M与非肌肉肌球蛋白IIA和IIB以及热休克蛋白70形成复合物，而C2 GnT-L与高尔基体磷蛋白3共定位。这些相互作用的性质尚不清楚。我们建议测试的假设，高尔基体定位的C2 GnT-L和C2 GnT-M是由它们的细胞质尾巴通过与特定的细胞质蛋白质的相互作用。本申请的具体目的是（1）鉴定与C2 GnT-M形成复合物的细胞质蛋白并表征复合物的结构;（2）鉴定与C2 GnT-L形成复合物的细胞质蛋白并表征复合物的结构;和（3）表征与C2 GnT-M和C2 GnT-M形成复合物的蛋白质的功能。L在控制这两种酶的细胞内运输和高尔基体定位以及粘蛋白糖基化中的作用。有和没有热休克的A549和H292细胞将是测试模型，因为它们表达C2 GnT-L、C2 GnT-M、MUC 1和MUC 5AC，这允许在破坏这些酶的高尔基体定位后评估这两种粘蛋白中粘蛋白聚糖结构的变化。将使用人支气管上皮细胞的原代培养物来确认这些细胞的结果。这些结果将扩大我们对糖基转移酶的细胞内运输和高尔基体保留机制的基本理解，并有助于为退伍军人的这两种肺部疾病制定治疗干预措施。 公共卫生相关性： 慢性阻塞性肺疾病和肺癌是许多退伍军人的两大医疗问题。这些患者的主要死因分别是粘液分泌过多和转移。这些疾病可能是由粘蛋白聚糖结构的改变引起的，其中分支结构发挥着关键作用。C2 GnT-M和C2 GnT-L控制分泌和膜结合粘蛋白中聚糖的分支结构，从而控制它们各自的功能。拟议的工作旨在了解这两种酶是如何定位到高尔基体，以及它们的细胞内运输和高尔基体定位的破坏如何影响粘蛋白聚糖的结构。研究结果将有助于了解这两种酶在肺部疾病和癌症中的作用，并可能有助于开发治疗这两种肺部疾病的治疗干预措施。

DESCRIPTION (provided by applicant): Chronic obstructive pulmonary diseases and lung cancer are two major medical problems for many veterans. The main causes of fatalities of these patients are mucus hypersecretion and metastasis, respectively. These diseases can result from alteration of mucin glycan synthesis and structure. Mucin-type glycans are elaborated in a stepwise manner from ser/thr as catalyzed by glycosyltransferases. Among them, branching enzymes are unique because they synthesize 26 N-acetylglucosaminide branch structures. These enzymes include core 2 N-acetylglucosaminyltransferase (C2GnT)-1/L, C2GnT-2/M, and C2GnT-3/T. C2GnT-L synthesizes core 2 in membrane glycoproteins and C2GnT-M synthesizes all three branch structures, core 2, core 4, and I antigen, in secreted mucins while the function of C2GnT-3 is unknown. Down regulation of C2GnT-L leads to defective immune function while overexpression causes cancer. Loss of function of C2GnT-M causes colon cancer. C2GnT-L and C2GnT-M will be the focus of this application. Glycan synthesis is determined by not only the amounts of glycosyltransferases but also their Golgi localization. The sub-Golgi localization of a glycosyltransferase closely matches the steps it participates in glycan synthesis. It is known that glycosyltransferases in colon cancer are mis-targeted, which results in the formation of short mucin glycans. The Golgi localization signal of glycosyltransferases resides mainly in the N-terminal cytoplasmic tail and transmembrane domain. However, the mechanism is not known. Our preliminary studies show that C2GnT-L and C2GnT-M are segregated intracellularly even though both synthesize core 2 and are expected to co- localize. In addition, C2GnT-M forms complexes with non-muscle myosin IIA & IIB and heat shock protein 70, while C2GnT-L co-localizes with Golgi Phosphoprotein 3. The nature of these interactions is not clear. We propose to test the hypothesis that Golgi localization of C2GnT-L and C2GnT-M is determined by their cytoplasmic tails via interaction with specific cytoplasmic proteins. The specific aims of this application are to (1) Identify the cytoplasmic proteins that form complexes with C2GnT-M and characterize the structure of the complexes; (2) Identify the cytoplasmic proteins that form complexes with C2GnT-L and characterize the structure of the complexes; and (3) Characterize the functions of the proteins that form complexes with C2GnT-M and C2GnT-L in controlling intracellular trafficking and Golgi localization of these two enzymes, and mucin glycosylation. A549 and H292 cells with and without heat shock will be the test models because they express C2GnT-L, C2GnT-M, MUC1 and MUC5AC, which allows assessment of changes in mucin glycan structures in these two mucins following disruption of Golgi localization of these enzymes. Primary cultures of human bronchial epithelial cells will be used to confirm the findings made with these cells. The results will expand our fundamental understanding of the mechanism of intracellular trafficking and Golgi retention of glycosyltransferases and could help develop therapeutic interventions for these two lung diseases in veterans. PUBLIC HEALTH RELEVANCE: Project Narrative Chronic obstructive pulmonary diseases and lung cancer are two major medical problems for many veterans. The main causes of the fatalities of these patients are mucus hypersecretion and metastasis, respectively. These diseases can result from alteration of mucin glycan structures in which branching structures play a key role. C2GnT-M and C2GnT-L control the branch structures of glycans in secreted and membrane-bound mucins and thus their respective functions. The proposed work aims at understanding how these two enzymes are localized to the Golgi and how disruption of their intracellular trafficking and Golgi localization could affect the structures of mucin glycans. The results will help understand the roles of these two enzymes in lung diseases and cancer, and may help develop therapeutic interventions for treating these two lung diseases.