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 支气管炎和哮喘，表现为高分泌和蓄积 呼吸道里有顽强的粘液。粘蛋白，一个主要的决定因素 粘液的流变性是由表面的分泌细胞分泌的 上皮和粘膜下腺。碳水化粘蛋白，它构成 80%-90%的分子是多相的，其结构可能是受调节的 由糖基转移酶的相对活性决定。粘蛋白核心2β6 G1cNAc 已知转移酶(Tf)和α2，6(或60)NeuAc Tf竞争 粘蛋白低聚糖的第一个分支点，产生低聚糖 具有不同的结构。我们假设相对的活动 分泌细胞中的这两种酶调节粘蛋白的酸性。 从这些细胞的不同组织化学染色中可以看出。它有 已经注意到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的。我们打算克隆 牛气管Beta6 G1cNAc Tf可形成核心2、核心4和I Beta6 G1cNAc结构和60NeuAc Tf的异种杂交 最近在其他动物物种中克隆了这两种酶的cDNA。我们 将通过鉴定受体的特异性来验证克隆的cDNA 这两种重组的水飞蓟素单抗(MAB) 这两个重组糖基转移因子将被生成并与{S- 用于探测牛气管上皮分泌的每种酶的核糖核酸探针 细胞分别进行免疫细胞化学和原位杂交。 这些结果将与组织化学应变特性相关联 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，以及优势是否 β6G1cNAc Tf与更多酸性粘蛋白的形成有关 含有更长的硫酸盐低聚糖和60NeuAc Tf 含较短唾液酸化粘蛋白的精制 低聚糖。同样的研究也将在原代细胞和 调节相对水平的牛气管上皮器官培养 通过反义寡核苷酸方法和基因方法对这两种酶的表达进行研究 腺病毒多聚赖氨酸结合物载体介导的转移 含有pCMV启动子。这些研究应该提供一个分子基础 在糖基转铁蛋白水平上了解 粘蛋白类碳水化合物的生物合成与分泌的异质性 健康和病变的呼吸道上皮细胞。