BIOSYNTHESIS OF TRACHEAL MUCOUS GLYCOPROTEINS

气管粘液糖蛋白的生物合成

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/2637987
• 关键词: antisense nucleic acid; bronchial mucus; carbohydrate structure; complementary DNA; glycoprotein biosynthesis; glycosyltransferase; goblet cells; histochemistry /cytochemistry; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; mucins; oligosaccharides; organ culture; polymerase chain reaction; respiratory epithelium; secretion; sialate; sulfation; tissue /cell culture; trachea

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.

慢性阻塞性肺疾病，如囊性纤维化、慢性阻塞性肺疾病 支气管炎和哮喘，表现为分泌过多和积聚 呼吸道中有顽固的粘液。 粘蛋白，一个重要的决定因素 粘液的流变特性，由表面的分泌细胞分泌 上皮和粘膜下腺。 粘蛋白碳水化合物，其组成 80-90% 的分子是异质的，其结构可以调节 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 alpha2,6（或 60）NeuAc TF 会竞争 粘蛋白寡糖的第一个分支点，产生寡糖 具有不同的结构。 我们假设相对活动 分泌细胞中的这两种酶调节粘蛋白的酸性特性 正如这些细胞的差异组织化学染色所反映的。 它有 值得注意的是，来自不同动物物种的相同糖基 TF 高度 氨基酸和核苷酸序列同源。 我们建议克隆 牛气管β6 G1cNAc TF，可形成核心2、核心4和I beta6 G1cNAc 结构和 60 NeuAc TF 通过跨物种杂交使用 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) 这两个重组糖基 TF 将生成并与 {S- 35}每种酶的核糖核酸探针探测牛气管上皮分泌 分别通过免疫细胞化学和原位杂交对细胞进行检测。 这些结果将与组织化学应变特性相关 这些细胞。 我们将确定这两个 TF 是否存在差异 在不同的分泌细胞群中表达，以及是否占优势 beta6G1cNAc TF 的形成与更多酸性粘蛋白的产生有关 含有较长的硫酸化低聚糖和 60 NeuAc TF 含有较短、唾液酸化的较少酸性粘蛋白的精制 寡糖。 同样的研究也将在原代细胞和 通过调节相对水平进行牛气管上皮的器官培养 通过反义寡核苷酸方法和基因检测这两种酶 使用载体由腺病毒聚赖氨酸缀合物介导的转移 含有pCMV启动子。 这些研究应该提供分子基础 在糖基 TF 水平上了解 粘蛋白型碳水化合物的生物合成和分泌的异质性 健康和患病气道上皮细胞。