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面临的两大医疗问题。导致这些患者死亡的主要原因分别是粘液高分泌和转移。这些疾病可能是由于粘蛋白多糖的合成和结构改变引起的。粘蛋白类型的多糖是在糖基转移酶的催化下，从Ser/Thr逐步形成的。其中，支链酶是独一无二的，因为它们合成了26个N-乙酰氨基葡萄糖支链结构。这些酶包括核心2 N-乙酰氨基葡萄糖转移酶(C2GnT)-1/L、C2GnT-2/M和C2GnT-3/T。C2GnT-L在膜糖蛋白中合成核心2，C2GnT-M在分泌的粘蛋白中合成核心2、核心4和I抗原，而C2GnT-3的功能尚不清楚。C2GnT-L表达下调导致免疫功能受损，过度表达则致癌。C2GnT-M功能丧失会导致结肠癌。C2GnT-L和C2GnT-M将是本申请的重点。糖链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定位与其参与糖合成的步骤非常匹配。已知在结肠癌中糖基转移酶被错误定位，从而导致短粘蛋白糖链的形成。糖基转移酶高尔基体定位信号主要位于N端胞质尾部和跨膜区。然而，其机制尚不清楚。我们的初步研究表明，C2GnT-L和C2GnT-M虽然都合成核心2，但都在细胞内分离，并有望共定位。此外，C2GnT-M与非肌肉肌球蛋白IIA&IIB和热休克蛋白70形成复合物，而C2GnT-L与高尔基体磷酸蛋白3共定位，这些相互作用的性质尚不清楚。我们提出了一种假设，即C2GnT-L和C2GnT-M的高尔基体定位是由它们的细胞质尾巴通过与特定的细胞质蛋白相互作用而决定的。本申请的具体目的是：(1)鉴定与C2GnT-M形成复合物的细胞质蛋白质并鉴定其结构；(2)鉴定与C2GnT-L形成复合物的细胞质蛋白质并鉴定其结构；以及(3)鉴定与C2GnT-M和C2GnT-L形成复合物的蛋白质在控制这两种酶的细胞内转运和高尔基定位以及粘蛋白糖基化方面的功能。A549和H292细胞在有和没有热休克的情况下都将是测试模型，因为它们表达C2GnT-L、C2GnT-M、MUC1和MUC5AC，这使得可以评估这些酶在高尔基体定位中断后这两种粘蛋白中粘蛋白多糖结构的变化。人支气管上皮细胞的原代培养将被用来证实用这些细胞所做的发现。这一结果将扩大我们对糖基转移酶细胞内转运和高尔基体滞留机制的基本理解，并有助于开发对这两种退伍军人肺部疾病的治疗干预措施。 公共卫生相关性： 项目简介慢性阻塞性肺疾病和肺癌是许多退伍军人面临的两大医疗问题。导致这些患者死亡的主要原因分别是粘液高分泌和转移。这些疾病可能是由于粘蛋白多糖结构的改变引起的，其中分支结构起着关键作用。C2GnT-M和C2GnT-L控制着分泌型和膜结合型粘蛋白中多糖的分支结构，从而控制着它们各自的功能。这项拟议的工作旨在了解这两种酶是如何定位到高尔基体上的，以及它们在细胞内的运输和高尔基体定位的中断如何影响粘蛋白多糖的结构。这一结果将有助于理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.