Biosynthesis of Tracheal Mucous Glycoproteins

气管粘液糖蛋白的生物合成

基本信息

批准号：
7528246
负责人：
PI-WAN CHENG
金额：
$ 46.87万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7528246
关键词：
Active Sites Affect Amino Acid Sequence Amino Acids Anabolism Anions Anodes Antibodies Asthma Binding Binding Proteins Biological Assay Biotin Blood typing procedure Breathing Carbohydrates Catalysis Cells Chinese Hamster Ovary Cell Chronic Bronchitis Colon Carcinoma Colorectal Cancer Complementary DNA Computer Graphics Computer software Conditioned Culture Media Coupled Crystallization Crystallography Cystic Fibrosis DNA DNA Polymerase I DNA Sequence Data Data Set Development Disaccharides Disease Down-Regulation Drops Electrophoresis Electrophoretic Mobility Shift Assay Enzymes Epidermal Growth Factor Epithelium Excision Exhibits Exons Fluorescence Funding Galactosidase Gel Gene Expression Gene Expression Regulation General Transcription Factors Genes Genomics Gland Glycoproteins Goals Goblet Cells Golgi Targeting Growth Hand Health Home environment Homologous Gene Housing Hyperplasia Hypertrophy Image In Vitro L-Selenomethionine Label LacZ Genes Length Luciferases Maps Measurement Measures Metals Methods Modeling Mucins Mucous body substance Mutagenesis Mutate Nuclear Extract Nucleotides Obstructive Lung Diseases Oils Peptide Signal Sequences Phase Plasmids Polysaccharides Positioning Attribute Procedures Property Protein Array Proteins R-factor RNA Interference Recombinant Proteins Regulation Regulatory Element Reporter Reporting Research Design Resolution Roentgen Rays Scanning Selenomethionine Signal Transduction Simulate Site Site-Directed Mutagenesis Sodium Chloride Solvents Source Streptavidin Structure Surface Synchrotrons System Testing Tissues Transfection Tretinoin Tumorigenicity Variant Water Weight X-Ray Crystallography base beta-1,3-Galactosyl-o-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase blood group cancer cell carbohydrate structure chromatin immunoprecipitation cytokine electron density enzyme activity evaporation fast protein liquid chromatography improved inhibitor/antagonist instrument magnetic beads malignant colon tumor mutant pathogen programs promoter receptor research study resistance factors synchrotron radiation therapy development three dimensional structure transcription factor vector

项目摘要

Mucus hypersecretion is a hallmark of obstructive lung diseases, including chronic bronchitis, asthma, and cystic fibrosis. This condition is the result of hypertrophy and hyperplasia of mucus cells. Secreted from goblet cells on the surface epithelium and mucus cells in the submucosal glands, mucins not only are the major determinant of the viscoelastic properties of mucus secretion but also can serve as the receptors for pathogens. The functions of mucins reside primarily in the carbohydrates, which constitute 70-90% of airway mucins by weight. In addition, mucin carbohydrates are very heterogeneous, which allow them to trap many different inhaled pathogens and facilitate their removal from the airways. Mucin carbohydrate structures and their functional potential can be expanded by core 2, core 4, and blood group I branch structures. All three structures can be formed by mucus tissue-specific core 2 N-acetylglucosaminyltransferase-M (C2GnT-M). Modulation of C2GnT-M gene expression can greatly affect the physicochemical properties of airway mucins and functions of airway mucus. Expression of C2GnT-M gene can be inhibited by epidermal growth factor but enhanced by retinoic acid and Th2 cytokines. C2GnT-M activity also can be regulated at the substrate level. Loss of C2GnT-M has been reported in colorectal cancer and its reexpression can inhibit tumorigenicity of colonic cancer cells. Thus, alteration of C2GnT-M can have a significant impact on health as well as diseases. The objective of this application is to characterize the modulation of C2GnT-M at the levels of enzyme activity and gene expression. We propose to: 1. Determine the active site of C2GnT-M by X-ray crystallography and then confirm the amino acids involved in catalysis by site-directed mutagenesis followed by measurement of enzyme activities using core 1, core 3, and blood group i disaccharide acceptors and their homologues. 2. Characterize C2GnT-M gene regulation by mapping cis-regulatory elements and identifying the cognate transcription factors under basal conditions. These transcription factors will be identified by transfection with cDNAs of known transcription factors and pull-down with biotinylated promoter followed by assay with transcription factor protein array. They will be characterized by electrophoresis mobility shift assay and chromatin immunoprecipitation assay. Current studies could facilitate the development of therapy for mucus hypersecretory diseases through identification of small carbohydrate inhibitors and mucus cell-specific promoter.

粘液分泌是阻塞性肺部疾病的标志，包括慢性支气管炎，哮喘和囊性纤维化。这种状况是粘液细胞肥大和增生的结果。粘液从表面上皮的杯状细胞和粘膜腺体的粘液细胞分泌，不仅是粘液分泌的粘弹性特性的主要决定因素，而且还可以用作病原体的受体。粘蛋白的功能主要驻留在碳水化合物中，碳水化合物构成了70-90％的气道粘蛋白。此外，粘蛋白碳水化合物非常异质，这使它们可以捕获许多不同的吸入病原体并促进其从气道中取出。粘蛋白碳水化合物结构及其功能潜力可以通过Core 2，Core 4和I群I分支结构扩展。所有三个结构都可以由粘液组织特异性核心2 N-乙酰葡萄糖氨基转移酶M（C2GNT-M）形成。 C2GNT-M基因表达的调节可以极大地影响气道粘蛋白的物理化学特性和气道粘液的功能。 C2GNT-M基因的表达可以被表皮生长因子抑制，但视黄酸和Th2细胞因子增强。 C2GNT-M活性也可以在底物水平上进行调节。已经报道了结直肠癌的C2GNT-M损失，其重新表达可以抑制结肠癌细胞的肿瘤性。因此，C2GNT-M的改变会对健康和疾病产生重大影响。该应用的目的是表征C2GNT-M在酶活性和基因表达水平上的调节。我们建议：1。通过X射线晶体学确定C2GNT-M的活性位点，然后确认通过位置定向的诱变涉及催化的氨基酸，然后使用CORE 1，CORE 3和IN CORE 3和IN CORE 3和血液组的二糖含量受体及其同源物测量酶活性。 2。通过绘制顺式调节元件并识别基础条件下的相关转录因子来表征C2GNT-M基因调节。这些转录因子将通过用已知转录因子的CDNA转染，并用生物素化启动子进行下拉，然后用转录因子蛋白阵列进行测定。它们的特征是电泳迁移率转移测定法和染色质免疫沉淀测定法。当前的研究可以通过鉴定小碳水化合物抑制剂和粘液细胞特异性启动子来促进粘液过度疾病的治疗。