MEMBRANE TOPOLOGY AND BIOSYNTHESIS OF GLYCOSAMINOGLYCANS

膜拓扑结构和糖胺聚糖的生物合成

• 批准号: 2177401
• 负责人: CARLOS Benjamin HIRSCHBERG
• 金额: $ 23.82万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177401
• 关键词: Golgi apparatus; affinity chromatography; carbohydrate biosynthesis; cell differentiation; complementary DNA; endoplasmic reticulum; enzyme mechanism; enzyme substrate; gel electrophoresis; gene expression; genetic library; hamsters; high performance liquid chromatography; immunoelectron microscopy; laboratory rat; liposomes; membrane activity; membrane permeability; membrane proteins; membrane structure; mitochondrial membrane; mucopolysaccharides; peptide chemical synthesis; protein biosynthesis; protein reconstitution; protein sequence; radionuclides; tissue /cell culture; transferase; transport proteins; uridine diphosphate glucuronate; xylose

The long-term goal of this research proposal is to understand whether there is regulation of the biosynthesis of different glycosaminoglycans through direct regulation of their biosynthetic enzymes (enzyme substrates and/or gene expression) and/or through membrane compartmentation of their specific substrates and enzymes. We hope that this will allow us an understanding of the regulation of the biosynthesis of proteoglycans under physiological and pathological conditions. Towards achieving this goal we will: (a) continue our studies on the N-heparan sulfate sulfotransferase, a key enzyme in the biosynthesis of heparan sulfate. Antibodies against the enzyme will be obtained and used to localize the enzyme within the Golgi apparatus by immunoelectromicroscopy. We will also clone its gene and express it in Chinese hamster ovary cell mutants deficient in this enzyme. This should allow one to understand the role of the enzyme and its product in many biological functions attributed to heparan sulfate proteoglycans. (b) Continue our studies on the purification of the Golgi adenosine 3' phosphate 5' phosphosulfate (PAPS) transporter. We will use our recently reconstituted PAPS transport assay into proteoliposomes to monitor the purification of the transporter. For this we will use and antibody against the carboxyatractyloside binding region of the transporter as well as 3' 5'-ADP-Sepharose affinity chromatography. Antibodies against the purified PAPS transporter will be used to study its sub-Golgi localization and its relationship with the N-heparan sulfate sulfotransferase within the Golgi membrane. (c) Continue with our studies on the ATP transporters of the Golgi apparatus and rough endoplasmic reticulum membrane. We will reconstitute these transporters into proteoliposomes and purify them by affinity chromatography. Antibodies against the transporters will be made to study their subcellular localization and the structural relationship among them as well as with the ATP/ADP transporter in mitochondria. (d) Purify the O-heparan sulfate sulfotransferase from rat liver Golgi membranes and prepare antibodies against the enzyme. These will be used to determine its subcellular localization, and whether or not it occurs within the membrane in a complex with the N-heparan sulfate sulfotransferase and the PAPS transporter. Concurrently, we will attempt to complement these studies with mutant Chinese hamster ovary cells deficient in this enzyme and the PAPS transporter. By expressing these genes in these mutants we should be able to understand the specific roles of proteoglycan sulfation in biological systems.

本研究计划的长期目标是了解是否存在 是通过调节不同糖胺聚糖的生物合成 直接调节其生物合成酶（酶底物和/或 基因表达）和/或通过其特定的膜区室 底物和酶。 我们希望这能让我们理解 生理条件下蛋白多糖生物合成的调控 和病理状况。 为了实现这一目标，我们将：(a) 继续我们对 N-硫酸乙酰肝素磺基转移酶的研究，这是一个关键的 硫酸乙酰肝素生物合成中的酶。 抗体针对 将获得酶并用于将酶定位在高尔基体内 免疫电子显微镜装置。 我们还将克隆它的基因并 在缺乏这种酶的中国仓鼠卵巢细胞突变体中表达它。 这应该能让人们了解酶及其产物的作用 许多生物学功能归因于硫酸乙酰肝素蛋白聚糖。 (b) 继续研究高尔基腺苷 3' 的纯化 磷酸盐 5' 磷酸硫酸盐 (PAPS) 转运蛋白。 我们将使用最近的 将 PAPS 转运测定重组到蛋白脂质体中以监测 转运蛋白的纯化。 为此，我们将使用抗 转运蛋白的羧基白术苷结合区以及 3' 5'-ADP-琼脂糖亲和层析。 纯化后的抗体 PAPS转运蛋白将用于研究其高尔基体下定位及其 与高尔基体内 N-硫酸乙酰肝素磺基转移酶的关系 膜。 (c) 继续我们对 ATP 转运蛋白的研究 高尔基体和粗面内质网膜。 我们将 将这些转运蛋白重组为脂蛋白体并通过以下方法纯化它们 亲和色谱法。 将制备针对转运蛋白的抗体 研究它们的亚细胞定位和结构关系 其中以及线粒体中的 ATP/ADP 转运蛋白。 (四) 从大鼠肝脏高尔基体中纯化O-硫酸乙酰肝素磺基转移酶 膜并制备抗该酶的抗体。 这些将用于 确定其亚细胞定位，以及它是否发生在 膜与 N-硫酸乙酰肝素磺基转移酶形成复合物， PAPS 转运蛋白。 同时，我们将尝试补充这些 对缺乏这种酶的突变中国仓鼠卵巢细胞的研究 和 PAPS 转运蛋白。 通过在这些突变体中表达这些基因，我们 应该能够理解蛋白多糖硫酸化的具体作用 在生物系统中。