BIOSYNTHESIS OF TRACHEAL MUCOUS GLYCOPROTEINS

气管粘液糖蛋白的生物合成

基本信息

批准号：
2637987
负责人：
PI-WAN CHENG
金额：
$ 23.04万
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 1999-12-31
项目状态：
已结题

项目摘要

Chronic obstructive pulmonary diseases, such as cystic fibrosis, chronic bronchitis and asthma, are manifested by hypersecretion and accumulation of tenacious mucus in the airways. Mucins, a major determinant of the rheological properties of mucus, are secreted by secretory cells in surface epithelium and submucosal glands. Mucin carbohydreate, which constitutes 80-90% of the molecule, is heterogeneous and its structure may be regulated by relative activities of glycosyltransferases. Mucin core 2 beta6 G1cNAc transferase (TF) and alpha2,6 (or 60) NeuAc TF are known to compete for the first branch point of mucin oligosaccharides, resulting in oligosaccharides with different structures. We hypothesize that relative activities of these two enzymes in the secretory cells regulate mucin acidic properties as reflected in differential histochemical staining of these cells. It has been noted that same glycosyl TF from different animal species is highly homologous in amino acid and nucleotide sequences. We propose to clone bovine tracheal beta6 G1cNAc TF, which can form core 2, core 4 and I beta6 G1cNAc structures, and 60 NeuAc TF by cross-species hybridization using recently cloned cDNAs of these two enzymes in other animal species. We will verify the cloned cDNAs by characterizing acceptor specificities of these two recombinant blycosyl TF's. Monoclonal antibodies (MABs) against these two recombinant glycosyl TFs will be generated and used with {S- 35}riboprobes for each enzyme to probe bovine tracheal epithelial secretory cells by immunocytochemistry and in situ hybridization, respectively. These results will be correlated with histochemical straining properties of these cells. We will ascertain whether these two TFs differentially express in different secretory cell populations, and whether predominance of beta6G1cNAc TF is associated with elaboration of more acidic mucins containing longer, sulfated olgosaccharides and 60 NeuAc TF with elaboration of less acidic mucins containing shorter, sialylated oligosaccharides. Same studies will also be performed in primary cell and organ cultures of bovine tracheal epithelium by modulating relative levels of these two enzymes by antisense oligonucleotide approach and by gene transfer mediated by an adenoviruspolylysine conjugate using a vector containing pCMV promoter. These studies should provide a molecular basis at the glycosyl TF level for understanding the regulation of the biosynthesis of mucin-type carbohydrates and the heterogeneity of secretory cells in healthy and diseased airway epithelium.

慢性阻塞性肺部疾病，例如囊性纤维化，慢性支气管炎和哮喘是由过度分泌和积累表现出来的气道顽强的粘液。粘蛋白，主要决定因素粘液的流变特性由表面分泌细胞分泌上皮和粘膜腺。构成的粘蛋白碳水化合物分子的80-90％是异质的，其结构可能受到调节通过糖基转移酶的相对活性。粘蛋白核心2 beta6 G1CNAC 已知转移酶（TF）和α2,6（或60）Neuac TF竞争粘蛋白寡糖的第一个分支点，导致寡糖具有不同的结构。我们假设分泌细胞中的这两种酶调节粘蛋白酸性特性如这些细胞的差异组织化学染色所反映。它有已经注意到，来自不同动物物种的相同糖基TF高度在氨基酸和核苷酸序列中同源。我们建议克隆牛气管Beta6 G1CNAC TF，可以形成核心2，核心4和我beta6 G1CNAC结构，使用跨物种杂交的60个NEUAC TF使用最近在其他动物物种中克隆了这两种酶的cDNA。我们将通过表征的受体特异性来验证克隆的cDNA 这两个重组blycosyl TF。单克隆抗体（mAb）将生成这两个重组糖基TF，并与{s-一起使用 35}每种酶的核糖探针探测牛气管上皮分泌通过免疫细胞化学和原位杂交细胞。这些结果将与组织化学特性相关这些细胞。我们将确定这两个TF是否有差异在不同的分泌细胞种群中表达 beta6g1cnac tf的阐述有关包含更长的，硫化的橄榄少ch糖和60个NEUAC TF 阐述含有较短，溶解的酸性粘蛋白较少的酸性粘蛋白寡糖。同样的研究也将在原代细胞和通过调节相对水平的牛气管上皮的器官培养在这两种酶中，通过反义寡核苷酸方法和基因通过使用载体的腺病毒胆碱结合物介导的转移包含PCMV启动子。这些研究应提供分子基础在糖基TF水平上，以了解调节粘蛋白型碳水化合物的生物合成和分泌的异质性健康和患病的气道上皮的细胞。