FEASIBILITY STUDY--STRUCTURAL/FUNCTIONAL DOMAINS OF LINK PROTEIN

可行性研究--连接蛋白的结构/功能域

• 批准号: 3769947
• 负责人: KAREN A MAGNUS
• 金额: --
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3769947
• 关键词: X ray crystallography; arthritis; biological models; cartilage; chemical structure; extracellular matrix; hyaluronate; intermolecular interaction; molecular cloning; protein structure function; proteoglycan; site directed mutagenesis

The major components of cartilage extracellular matrix consist of collagen, fibrils, proteoglycans and water. The chondroitin sulfate proteoglycan (aggrecan) is comprised of proteoglycan core protein substituted with multiple chains of chrondroitin sulfate and keratan sulfate. The proteoglycan complex is, in turn, non-covalently bound to hyaluronate, interactions which are stabilized by a third component, the link protein. Link protein and the aggregating proteoglycan are related macromolecules, showing significant primary sequence homology in regions with similar function. Hyaluronate binding domains in the link protein and proteoglycan are also related to domains found in the lymphocyte homing receptor (CD44). Detailed structural information about the molecular interactions between these components of the proteoglycan aggregate of cartilage upon which cartilage structure and function depend, will enhance our understanding of cartilage biochemistry in normal and disease states such as arthritis and structural information concerning functional domains of the link protein molecule will related to the homologous functionally similar domains in the aggrecan monomer. Furthermore, these studies may help to explain the functional significance of homologous proteins on lymphocyte cell surfaces. The atomic resolution structure of link protein will be studied using a combination of molecular biological, biochemical and x-ray crystallographic techniques. Over the three years of the proposed grant period we will obtain crystals suitable for three dimensional atomic resolution studies of the intact link protein and isolated structural domains produced using recombinant DNA techniques. Once appropriate crystals of link protein are available structure analysis by single crystal x-ray diffraction methods will be undertaken. Finally the resultant structures will be examined in light of their known functional roles. This development and feasibility study will provide the basis for ongoing collaborative studies between the principal investigators. The information obtained will be used to initiate more comprehensive investigations into cartilage extracellular matrix macromolecular structure, as well as to influence ongoing studies to determine sites of functional interactions between components of the proteoglycan aggregate. Related studies already in progress involve cloning, site directed mutagenesis and functional determinations of link protein and aggrecan domains. Three dimensional atomic resolution structural information once obtained will enable the rational design of mutant forms of link protein that can be produced by recombinant DNA technology, studied using crystallographic techniques and assessed for function by well established biochemical methods.

软骨细胞外基质的主要成分包括 胶原、原纤维、蛋白聚糖和水。 硫酸软骨素 蛋白聚糖（聚集蛋白聚糖）由蛋白聚糖核心蛋白组成 被硫酸软骨素和角质素的多链取代 硫酸盐 蛋白聚糖复合物又非共价结合至 透明质酸盐，通过第三组分稳定的相互作用， 连接蛋白 连接蛋白与聚集蛋白聚糖有关 大分子，在区域中显示出显著的一级序列同源性 类似的功能。 连接蛋白中的透明质酸结合结构域 和蛋白聚糖也与淋巴细胞中发现的结构域有关 归巢受体（CD44）。 详细的结构信息 蛋白聚糖的这些组分之间的分子相互作用 软骨结构和功能所依赖的软骨集合体 依赖，将提高我们对软骨生物化学的理解， 正常和疾病状态，如关节炎和结构信息 关于连接蛋白分子的功能结构域， 与聚集蛋白聚糖单体中同源的功能相似的结构域。 此外，这些研究可能有助于解释功能性 淋巴细胞表面同源蛋白的意义。 的 连接蛋白的原子分辨率结构将使用 分子生物学、生物化学和X射线的结合 晶体学技术 在拟议拨款的三年内， 在此期间，我们将获得适合于三维原子的晶体。 完整连接蛋白和分离结构的解析研究 使用重组DNA技术产生的结构域。 一旦合适 连接蛋白的晶体可通过单克隆抗体进行结构分析。 将采用晶体x射线衍射方法。 最后 所得结构将根据其已知的功能进行检查， 角色 这项开发和可行性研究将为以下方面提供基础： 主要研究者之间正在进行的合作研究。 的 获得的信息将用于启动更全面的 软骨细胞外基质大分子研究进展 结构，以及影响正在进行的研究，以确定网站的 蛋白聚糖聚集体组分之间的功能性相互作用。 相关的研究已经在进行中，包括克隆、定点定向 连接蛋白和聚集蛋白聚糖诱变和功能测定 域. 三维原子分辨率结构信息一次 将使合理设计突变形式的连接蛋白 可以通过重组DNA技术生产，使用 晶体学技术，并通过公认的 生物化学方法