Glycosyltransferase Golgi Retention Mechanism

糖基转移酶高尔基体保留机制

• 批准号: 8598013
• 负责人: PI-WAN CHENG
• 金额: --
• 依托单位: OMAHA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598013
• 关键词: A549; Acetylgalactosamine; Binding; Breathing; C2GnT-mucin; Cancer Etiology; Carbohydrates; Cells; Chronic Obstructive Airway Disease; Colitis; Colon Carcinoma; Complex; Cytoplasmic Protein; Cytoplasmic Tail; Development; Disease; Down-Regulation; Enzymes; Epithelial Cells; Epithelium; Gene Deletion; Genes; Golgi Apparatus; Heat-Shock Proteins 70; Heat-Shock Response; Human; I-antigen; Injury; Isoenzymes; Knockout Mice; Lead; Leukocyte Trafficking; Link; Lung diseases; Lymphoid Tissue; MUC5AC gene; Malignant neoplasm of lung; Medical; Membrane; Membrane Glycoproteins; Minor; Modeling; Mucin-1 Staining Method; Mucins; Mucous body substance; N-terminal; Natural Immunity; Nature; Neoplasm Metastasis; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Patients; Phosphoproteins; Polysaccharides; Proteins; Selectins; Signal Transduction; Site; Structure; Tandem Repeat Sequences; Testing; Therapeutic Intervention; Thymus Gland; Tissues; Transmembrane Domain; Up-Regulation; Veterans; Water; acquired immunity; airway obstruction; beta-1,3-Galactosyl-o-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase; glycosylation; glycosyltransferase; immune function; leukocyte activation; loss of function; mouse model; non-muscle myosin; overexpression; pathogen; protein aminoacid sequence; protein function; sialyl Lewis x; therapy development; trafficking

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.

描述（由申请人提供）： 慢性阻塞性肺病和肺癌是许多退伍军人面临的两大健康问题。这些患者死亡的主要原因分别是粘液分泌过多和转移。这些疾病可能是由于粘蛋白聚糖合成和结构的改变引起的。粘蛋白型聚糖是在糖基转移酶的催化下从丝氨酸/苏氨酸逐步加工而成。其中，分支酶是独特的，因为它们合成26个N-乙酰氨基葡萄糖分支结构。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所有三个分支结构：核心 2、核心 4 和 I 抗原，而 C2GnT-3 的功能未知。 C2GnT-L 的下调会导致免疫功能缺陷，而过度表达则会导致癌症。 C2GnT-M 功能丧失会导致结肠癌。 C2GnT-L和C2GnT-M将是本次应用的重点。聚糖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。糖基转移酶的高尔基体下定位与其参与聚糖合成的步骤密切匹配。众所周知，结肠癌中的糖基转移酶定位错误，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端胞质尾部和跨膜结构域。然而，其机制尚不清楚。我们的初步研究表明，C2GnT-L 和 C2GnT-M 在细胞内分离，尽管两者都合成核心 2 并且预计会共定位。此外，C2GnT-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 C2GnT-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。我们建议检验以下假设：C2GnT-L 和 C2GnT-M 的高尔基体定位是由它们的细胞质尾部通过与特定细胞质蛋白的相互作用决定的。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并表征复合物的结构； (2) 鉴定与C2GnT-L形成复合物的细胞质蛋白并表征复合物的结构； (3) 表征与 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运输和高尔基体定位以及粘蛋白糖基化中的功能。有和没有热休克的 A549 和 H292 细胞将成为测试模型，因为它们表达 C2GnT-L、C2GnT-M、MUC1 和 MUC5AC，这允许评估这两种粘蛋白中的粘蛋白聚糖结构在这些酶的高尔基体定位被破坏后的变化。人支气管上皮细胞的原代培养物将用于证实这些细胞的研究结果。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，并有助于开发针对退伍军人的这两种肺部疾病的治疗干预措施。