Biosynthesis of Tracheal Mucous Glycoproteins

粘液分泌过多是阻塞性肺疾病的标志，包括慢性支气管炎、哮喘和囊性纤维化。这种情况是粘液细胞肥大和增生的结果。粘蛋白由表面上皮的杯状细胞和粘膜下腺的粘液细胞分泌，不仅是粘液分泌粘弹性的主要决定因素，而且可以作为病原体的受体。粘蛋白的功能主要存在于碳水化合物中，碳水化合物占气道粘蛋白重量的70-90%。此外，粘蛋白碳水化合物是非常异质的，这使它们能够捕获许多不同的吸入病原体，并促进它们从气道中清除。黏液蛋白碳水化合物结构及其功能潜力可通过核心2、核心4和血型I分支结构扩展。这三种结构均可由黏液组织特异性核心2 n -乙酰氨基葡萄糖转移酶- m （C2GnT-M）形成。C2GnT-M基因表达的调节对气道黏蛋白的理化性质和气道黏液的功能有很大的影响。表皮生长因子可抑制C2GnT-M基因的表达，而视黄酸和Th2细胞因子可增强C2GnT-M基因的表达。C2GnT-M活性也可以在底物水平上调节。C2GnT-M的缺失在结直肠癌中有报道，其再表达可以抑制结肠癌细胞的致瘤性。因此，C2GnT-M的改变可以对健康和疾病产生重大影响。本应用的目的是表征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，然后通过位点定向诱变确定参与催化的氨基酸，随后使用核心1、核心3和i型血双糖受体及其同源物测量酶活性。2. 在基础条件下，通过定位顺式调控元件和鉴定同源转录因子来表征C2GnT-M基因调控。这些转录因子将通过转染已知转录因子的cdna，用生物素化启动子拉下，然后用转录因子蛋白阵列检测来鉴定。它们将通过电泳迁移迁移试验和染色质免疫沉淀试验进行表征。目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高分泌性疾病的治疗发展。

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.