GLYCOPROTEIN PROCESSING SYSTEMS OF ER AND GOLGI

本申请涉及糖基化和糖蛋白 处理. 相关酶定位于内质网 内质网（ER）和高尔基体。 在糖基化领域， 我们的实验室一直在研究地形学、组织学和 单个酶的作用机制。 未来的具体目标 资助期间包括继续研究多里科尔的地形 磷酸甘露糖合成酶，长醇，克隆及序列分析 磷酸葡萄糖合成酶，并进一步探索了一个13个氨基酸 ER糖基转移酶中的肽序列，其可作为识别 聚类异戊二烯化合物磷酸多萜醇的位置。 其他ER 项目包括分离和测序Dol-P-Man合酶cDNA， 哺乳动物组织和酵母ALG 3基因的克隆， 编码一种催化长醇跨膜运动的酶 焦磷酸连接的寡糖。 在平行于这个程序在糖基化，一个程序有关的基本 糖蛋白的作用机制正在研究中。 在酵母的帮助下 分子生物学，目前的模型，从核苷酸糖运输 细胞质转化为高尔基体囊泡的实验正在进行中。 我们也开始 克隆和表征酵母高尔基甘露糖基转移酶。 哺乳动物高尔基体加工甘露聚糖酶基因的克隆和序列分析 这是另一个重点。 甘露糖苷酶II的测序具有 最近完成，并与约翰舒茨巴赫博士合作，我们 已经将该程序扩展到肝α 1，2-甘露糖苷酶I的克隆。 将尝试在酵母中表达两种哺乳动物甘露聚糖酶， 确定功能性高尔基体定位信号是否在 酵母和哺乳动物。 最后，与福田康夫博士合作， 将调查一个遗传性疾病HEMPAS的病例。 初步数据 表明这种情况可能与低于正常水平的 甘露糖苷酶II。

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.