Biosynthesis of Tracheal Mucous Glycoproteins

粘液高分泌是阻塞性肺疾病的特征，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殖的结果。粘蛋白由表面上皮上的杯状细胞和粘膜下腺中的粘液细胞分泌，不仅是粘液分泌粘弹性的主要决定因素，也是病原体的受体。粘蛋白的功能主要存在于碳水化合物中，按重量计算，碳水化合物占呼吸道粘蛋白的70%-90%。此外，粘蛋白碳水化合物是非常不同的，这使得它们能够捕获许多不同的吸入病原体，并促进它们从呼吸道中清除。粘蛋白碳水化合物结构及其功能潜力可以通过核心2、核心4和血型I分支结构来扩展。这三种结构都可以由粘液组织特异性核心2 N-乙酰氨基葡萄糖基转移酶-M(C2GnT-M)形成。C2GnT-M基因表达的调控可极大地影响呼吸道粘蛋白的理化性质和呼吸道粘液功能。表皮生长因子可抑制C2GnT-M基因的表达，维甲酸和Th2细胞因子可促进C2GnT-M基因的表达。C2GnT-M活性也可以在底物水平上进行调节。C2GnT-M的缺失在结直肠癌中已有报道，其再表达可抑制结肠癌细胞的致瘤性。因此，C2GnT-M的改变对健康和疾病都有重大影响。本应用的目的是在酶活性和基因表达水平上表征C2GnT-M的调控。我们建议：1.通过X射线结晶学确定C2GnT-M的活性部位，然后通过定点突变确定参与催化的氨基酸，然后利用核心1、核心3和血型I双糖受体及其同系物测定酶活性。2.在基础条件下定位顺式调控元件，鉴定同源转录因子，研究C2GnT-M基因的调控。这些转录因子将通过已知转录因子的cDNA转染和生物素化启动子的下拉，然后用转录因子蛋白质阵列进行检测来鉴定。它们将通过电泳法、迁移率改变法和染色质免疫沉淀法进行表征。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。

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.