CHONDROITIN SULFATE BIOSYNTHESIS CELLULAR LOCALIZATION

硫酸软骨素生物合成细胞定位

• 批准号: 2080867
• 负责人: GEETHA SUGUMARAN
• 金额: $ 11.8万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2080867
• 关键词: Golgi apparatus; affinity chromatography; animal tissue; carbohydrate biosynthesis; chondroitin sulfates; enzyme complex; enzyme mechanism; glycoprotein biosynthesis; glycosyltransferase; intracellular transport; mucopolysaccharides; protein purification; proteoglycan; sulfotransferase

Proteochondroitin sulfate is a major constituent of the cartilage matrix and all other connective tissue. It plays an important role in bone and connective tissue metabolism, wound healing, and in cell interactions. Defective synthesis of these molecules could impact upon diseases and conditions such as arthritis, atherosclerosis, improper wound healing, etc. Better knowledge of the biosynthesis and its regulation may assist in providing rational therapies for these conditions. Proteochondroitin sulfates consist of a protein backbone to which many glycosaminoglycan chains of varying chain length and sulfate content are covalently attached. The chondroitin chains are linked to a wide range of core protein families. The functions of these proteoglycans are not clearly defined, but may be as varied as their structures. It is unclear how the protein core and the highly anionic sulfated glycosaminoglycan structures direct these functions, but it is likely that the core protein would be involved in channeling the transport and presentation of the attached glycosaminoglycans to specific intracellular granule, cell surface, or matrix localities. Whether or not this is a main function of the protein core, it does seem apparent that the fine structure of the highly anionic glycosaminoglycans may be of paramount importance in function at each location where the proteoglycans are presented. It is not clear at present as to what key features control the biosynthesis of the fine structure of the chondroitin sulfate chains. Our goal is to examine the assembly of the polysaccharide portion of proteochondroitin sulfate. Although the main outlines of the biosynthetic pathway are now clear, several areas remain to be explored. These include the organization, sub-cellular localization, movement and interaction of nascent proteoglycan and enzyme for synthesis of the polysaccharide portion of proteochondroitin sulfate. We will use a chick embryo epiphyseal cartilage system that synthesizes at least two populations of proteochondroitins that are predominantly 6-sulfated and a mast cell system that synthesizes an intracellular proteochondroitin 4-sulfate. In order to examine the organization of the enzymes involved in synthesis, we will sub-fractionate the Golgi followed by functional characterization of the fractions. These fractions will be assayed for xylosyl transferase, two galactosyl transferases, N-acetylgalactosaminyl transferase(s), glucuronosyl transferase(s), 4-sulfotransferase, 6- sulfotransferase and specific marker enzymes. In addition, N- acetylgalactosaminyl transferase, glucuronosyl transferase and sulfotransferases will be purified to homogeneity, employing conventional protein purification techniques as well as affinity chromatographic techniques. The fundamental characteristics and specificities of these enzymes will be determined. The interactions of the enzymes with each other to form multi-enzyme complexes will be examined. This will provide the basis for the long-term goal which is to reconstitute proteochondroitin sulfate synthesis with the purified enzymes, raise antibodies to the purified enzymes and to clone them.

硫酸蛋白软骨素是软骨基质的主要成分 以及所有其他结缔组织。 它在骨头和 结缔组织代谢，伤口愈合和细胞相互作用。 这些分子的缺陷综合可能会影响疾病，并 诸如关节炎，动脉粥样硬化，伤口愈合不当之类的疾病， 等。对生物合成及其调节的更好了解可能有助于 为这些条件提供合理疗法。 蛋白软骨素硫酸盐由蛋白质主链组成，其中许多 不同链长和硫酸盐含量的糖胺聚糖链是 共价附加。 软骨素链与广泛的范围有关 核心蛋白质家族。 这些蛋白聚糖的功能不是 明确定义，但可能与它们的结构一样多样。 这是 尚不清楚蛋白质核和高阴离子硫酸如何 糖胺聚糖结构指导这些功能，但很可能是 核心蛋白将参与引导运输和 将附着的糖胺聚糖介绍给特定 细胞内颗粒，细胞表面或基质部位。 是否或 这不是蛋白质核心的主要功能，看起来确实很明显 高度阴离子糖胺聚糖的精细结构可能是 在功能中的功能至关重要的每个位置 提出了蛋白聚糖。 目前尚不清楚什么钥匙 特征控制着精细结构的生物合成 硫酸软骨素链。 我们的目标是检查多糖部分的组装 硫酸蛋白软骨素。 虽然 生物合成途径现在很清楚，仍有几个区域待探索。 这些包括组织，细胞局部定位，运动和 新生蛋白聚糖和酶的相互作用以合成 硫酸蛋白石头骨素的多糖部分。 我们将使用一个 至少合成两个 主要是6种阳离子的蛋白质软骨素的种群 合成细胞内蛋白软骨素的肥大细胞系统 4-硫酸盐。 为了检查涉及的酶的组织 合成中，我们将对高尔基体进行亚分数，然后进行功能 分数的特征。 这些分数将被分析 木糖基转移酶，两个半乳糖基转移酶，N-乙酰乳糖胺基因 转移酶，葡萄糖基转移酶，4-硫代转移酶，6- 亚磺转移酶和特定标记酶。 另外，n- 乙酰乳糖苷基因转移酶，葡萄糖醛酸转移酶和 硫代转移酶将被纯化为均匀性 常规蛋白质纯化技术以及亲和力 色谱技术。 基本特征和 这些酶的特异性将被确定。 相互作用 酶相互形成多酶复合物将是 检查。 这将为长期目标提供基础 用纯化的 酶，对纯化的酶产生抗体并克隆它们。