GLYCOPROTEIN PROCESSING SYSTEMS OF ER AND GOLGI

内质网和高尔基体的糖蛋白加工系统

• 批准号: 3304555
• 负责人: PHILLIPS W ROBBINS
• 金额: $ 24.11万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304555
• 关键词: Golgi apparatus; Saccharomyces; carbohydrate transport; cell membrane; chimeric proteins; cytoplasm; dolichol; endoplasmic reticulum; enzyme structure; glycoprotein biosynthesis; glycosidases; glycosylation; glycosyltransferase; inborn carbohydrate metabolism disorder; laboratory rabbit; mannose; mannosidase; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid sequence; phosphorylation; polymerase chain reaction; posttranslational modifications; protein purification; protein sequence; protein structure function; site directed mutagenesis; southern blotting; sugar nucleotide; tissue /cell culture

This application is concerned with glycosylation and glycoprotein processing. The relevant enzymes are localized in the endoplasmic reticulum (ER) and Golgi. In the glycosylation area, recent emphasis in our laboratory has been on basic questions of topography, organization, and mechanism of action of individual enzymes. Specific aims for the coming grant period include continued study of the topography of dolichol phosphate mannose synthase, the cloning and sequencing of dolichol phosphate glucose synthase, and further exploration of a 13 amino acid peptide sequence in ER glycosyltransferases that may serve as a recognition site for the polyisoprenoid compound, dolichol phosphate. Other ER projects include isolation and sequencing of Dol-P-Man synthase cDNAs from mammalian tissues and the cloning of the yeast ALG3 gene, a gene that may encode an enzyme which catalyzes transmembrane movement of dolichol pyrophosphate linked oligosaccharides. In parallel with this program in glycosylation, a program concerning basic mechanisms of glycoprotein is being developed. With the help of yeast molecular biology, current models for nucleotide sugar transport from the cytoplasm into Golgi vesicles are being tested. We are also beginning to clone and characterize yeast Golgi mannosyl transferases. The cloning and sequencing of mammalian Golgi processing mannosidases represents another major emphasis. The sequencing of mannosidase II has recently been completed and in collaboration with Dr. John Schutzbach we have extended this program to the cloning of liver alpha1,2-mannosidase I. Attempts will be made to express both mammalian mannosidases in yeast to determine whether functional Golgi localization signals are shared between yeast and mammals. Finally, in collaboration with Dr. Michiko Fukuda we will investigate a case of the genetic disease HEMPAS. Preliminary data indicate that this case may be associated with lower than normal levels of mannosidase II.

该应用与糖基化和糖蛋白有关 加工。 相关酶位于内质中 网状（ER）和高尔基大学。 在糖基化区域，最近重点 我们的实验室一直在地形，组织和 单个酶的作用机理。 到来的具体目标 赠款期包括继续研究多利希尔的地形 磷酸甘露糖合酶，多利果醇的克隆和测序 磷酸盐葡萄糖合酶，并进一步探索13个氨基酸 ER糖基转移酶中的肽序列，可以用作识别 聚异型化合物Dolichol磷酸盐的位点。 其他ER 项目包括隔离和从DOL-P-MAN合成酶CDNA的测序 哺乳动物组织和酵母Alg3基因的克隆，该基因可能 编码一种催化Dolichol跨膜运动的酶 焦磷酸连接的寡糖。 与糖基化中的该程序并行，一个有关基本的程序 正在开发糖蛋白的机制。 在酵母的帮助下 分子生物学，当前的核苷酸糖转运模型 正在测试细胞质成高尔基体囊泡。 我们也开始 克隆和表征酵母高尔基甘露糖基转移酶。 哺乳动物高尔尔基加工甘露糖苷酶的克隆和测序 代表另一个主要重点。 甘露糖苷酶II的测序有 最近完成并与John Schutzbach博士合作 已将该程序扩展到肝α1,2-甘露糖苷酶的克隆。 将尝试在酵母中表达两种哺乳动物甘露糖苷酶 确定功能性高尔基体定位信号是否共享 酵母和哺乳动物。 最后，与Michiko Fukuda博士合作 将研究遗传疾病大麻的病例。 初步数据 表明这种情况可能与低于正常水平相关 甘露糖苷酶II。