GLYCOSYLTRANSFERASES, MUCINS AND MACROPHAGE RECEPTORS

糖基转移酶、粘蛋白和巨噬细胞受体

• 批准号: 2174529
• 负责人: ROBERT L HILL
• 金额: $ 61.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174529
• 关键词: Kupffer's cell; affinity chromatography; antibody; carbohydrate biosynthesis; carbohydrate structure; chemical binding; complementary DNA; enzyme structure; genetic library; glucose receptor; glycoproteins; glycosyltransferase; growth /development; high performance liquid chromatography; immunoelectron microscopy; laboratory mouse; laboratory rabbit; laboratory rat; macrophage; molecular cloning; mucins; nucleic acid sequence; oligosaccharides; point mutation; protein engineering; protein sequence; protein structure function; submandibular gland; swine; tissue /cell culture; transposon /insertion element

The structure-function relationships of three groups of proteins that are involved in aspects of glycoconjugate biosynthesis or function will be examined. GLYCOSYLTRANSFERASES that act in the biosynthesis of oligosaccharides in glycoconjugates will be studied in several ways. 1) cDNA expression libraries in lambda gtll will be screened with antibodies to certain transferases and clones containing transferase cDNA will be isolated and sequenced. 2) The cDNA for certain transferases will be inserted into appropriate expression vectors to obtain sufficient transferase for structure/function studies or to obtain specifically modified enzyme by site specific mutagenesis. 3) Two transferases that act in mucin oligosaccharide biosynthesis and have resisted purification will be purified and characterized. The two transferases form the core sequence GAl beta 1,3Ga1NAc-O- Ser/Thr. MUCINS that serve to lubricate the gastrointestinal, respiratory and urogenital tracts and to protect epithelial cells from injury and dehydration will be isolated and further characterized, as follows. 1) The cDNA sequence of porcine submaxillary gland cDNA will be completed and the sequence of genomic DNA for submaxillary mucin examined. In addition, the number of genes for mucin in the porcine genome will be determined. 2) Antibodies to mucin and synthetic polynucleotides based on submaxillary mucin cDNA sequence will be used to determine whether other porcine tissues contain submaxillary mucin-like protein or mRNA. It will also be determined with use of these probes whether human submaxillary apomucin is structurally related to porcine submaxillary apomucin. 3) The exact location of oligosaccharides in mucin will be determined. 4) Mucins from other porcine tissues, such as stomach, small intestine and colon will be isolated and structurally characterized. MACROPHAGE RECEPTORS for oligosaccharides will be examined, especially the receptor found only on rat and mouse Kupffer cells. 1) The sequence of the genomic DNA for rat Kupffer cells will be determined. 2) The expression of the receptor during macrophage development will be examined and a search for the exact function of the receptor will be made. 3) The structural basis for the binding of carbohydrate to the receptor will be sought.

三组蛋白质的结构-功能关系 涉及糖共轭生物合成或 功能将被检查。糖基转移酶起作用 在低聚糖的生物合成中糖共轭化合物将是 从几个方面进行了研究。1)在lambda中构建的表达文库 将用某些转移酶的抗体和 将分离含有转移酶基因的克隆，并 已排序。2)插入某些转移酶的cdna 转化成适当的表达载体，以获得足够的 用于结构/功能研究的转移酶或获得特定的 定点突变修饰酶。3)两个 参与粘蛋白低聚糖生物合成的转移酶 已经抵抗提纯的将被提纯和鉴定。这个 两个转移酶形成核心序列Galβ1，3Ga1NAc-O- 系列/三。用来润滑胃肠道的粘蛋白， 呼吸道和泌尿生殖道，并保护上皮细胞 从受伤和脱水中分离出来，并进一步 其特征如下。1)猪的cdna序列 将完成颌下腺基因的克隆和测序 对颌下粘液进行基因组DNA检测。此外， 猪基因组中粘蛋白的基因数量将是 下定决心。2)抗粘蛋白和合成多核苷酸抗体 根据颌下粘蛋白的cdna序列，将其用于 确定其他猪组织是否含有下颌骨 粘蛋白样蛋白或信使核糖核酸。它也将通过使用来确定 在这些探针中，人类颌下无粘蛋白是否 在结构上与猪上颌下无粘蛋白有亲缘关系。3) 低聚糖在粘蛋白中的确切位置将被确定。4) 来自其他猪组织的粘蛋白，如胃，小的 肠道和结肠将被分离，并具有结构特征。 巨噬细胞低聚糖受体将是 被检查的，尤指仅在大鼠和小鼠身上发现的受体 库普弗细胞。1)大鼠基因组DNA序列 库普弗细胞将被确定。2)表达 巨噬细胞发育过程中的受体将被检测 将对受体的确切功能进行研究。3) 碳水化合物与糖链结合的结构基础 将寻找受体。