INTERMOLECULAR PHYSICAL INTERACTIONS IN CARTILAGE

软骨中的分子间物理相互作用

• 批准号: 6375178
• 负责人: Steven J Eppell
• 金额: $ 10.57万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375178
• 关键词: SDS polyacrylamide gel electrophoresis; atomic force microscopy; biomechanics; cartilage; chondroitin sulfates; collagen; conformation; density gradient ultracentrifugation; extracellular matrix; image processing; intermolecular interaction; ionic bond; mechanical pressure; osteoarthritis; pathologic process; protein purification; proteoglycan; viscosity

The long-term objective of this research effort is to find preventative and/or curative measures for osteoarthritis by enhancing understanding of the molecular and sub-molecular scale pathogenesis of this disease. The central hypothesis of this proposal is that the location and magnitude of submolecularly localized charge domains on type IX collagen are important in the interaction between collagen fibrils and aggrecan molecules in the cartilagenous extracellular matrix (ECM). Type II collagen fibrils are hypothesized to link to the aggregated proteoglycan matrix via an electrostatic interaction between negatively charged sulfate groups on aggrecan glycosaminoglycan side chains and the theoretical highly positively charged non-collagenous 4 (NC4) domain of type IX collagen which is covalently bound to the surface of the type II fibrils. There is substantial circumstantial evidence that this interaction is crucial in obtaining and maintaining normal viscoelastic biomechanical function of the ECM. The work proposed will provide a direct measurement of this interaction. In addition, in vitro sub- molecular scale measurements of the conformation and charge distribution of purified monomers of type II and type IX collagen and aggrecan will be obtained. This information will be critical in the development of the next generation of molecularly-based theoretical models of ECM biomechanics. The Specific Aims of the proposal are to: 1) image individual surface resident type II and type IX collagen and aggrecan molecules under ambient and in vitro conditions; 2) measure the force fields generated by each of the molecules; 3) measure the force of interaction between chondroitin sulfate (CS) and type II and type IX collagen; and 4) measure the force of interaction between aggrecan and type II and type IX collagen molecules. These aims will be accomplished using an atomic force microscope to probe molecules immobilized on a surface. By analyzing data in the snap-to-contact portion of a standard force-distance curve, the magnitude of charges on the collagen and aggrecan molecules will be obtained (Aim 2). The small size of the end of the AFM probe tip allows for this measurement to be made with sub- molecular spatial resolution. Aims 3 and 4 involve covalent attachment of CS and aggrecan to an AFM probe tip. Subsequent analysis of the adhesive region of the force-distance curve will yield the forces of interaction. The applicant suggests that results of the proposal experiments will unequivocally answer the question of whether type IX collagen is capable of participating in an electrostatic interaction with aggrecan that is strong enough to hold cartilage together under physiological conditions.

这项研究工作的长期目标是寻找预防性 和/或通过增强理解的骨关节炎的治疗方法 该疾病的分子和亚分子尺度发病机理。 该提议的中心假设是位置和 IX型胶原蛋白上的分子局部电荷域的大小 在胶原纤维和脂肪蛋白之间的相互作用中很重要 软骨细胞外基质（ECM）中的分子。 II型 假设胶原蛋白原纤维链接到聚集的蛋白聚糖 通过负电荷之间的静电相互作用矩阵 脂肪酸酯糖胺聚糖侧链的硫酸盐基团和 理论高度充电的非胶原4（NC4）域 IX类型胶原蛋白，共价结合到该类型的表面 II原纤维。 有大量的间接证据表明这 相互作用对于获得和维持正常粘弹性至关重要 ECM的生物力学功能。 提议的工作将提供 直接测量这种相互作用。 另外，体外亚 - 构象和电荷分布的分子尺度测量 II型和IX型胶原蛋白和Aggrecan的纯化单体将 获得。 这些信息对于发展至关重要 ECM的下一代基于分子的理论模型 生物力学。 该提案的具体目的是：1）图像 单个表面居民II型和IX型胶原蛋白和Aggrecan 分子在环境和体外条件下； 2）测量力 每个分子产生的磁场； 3）测量的力 硫酸软骨素（CS）与II型和IX型之间的相互作用 胶原; 4）衡量Aggrecan和Aggrecan之间的相互作用力 II型和IX型胶原蛋白分子。 这些目标将实现 使用原子力显微镜探测固定在A上的分子 表面。 通过分析标准的快速接触部分中的数据 力距离曲线，胶原蛋白上的电荷大小 将获得Aggrecan分子（AIM 2）。末端的小尺寸 AFM探针尖端允许通过子进行此测量 分子空间分辨率。目标3和4涉及共价附件 CS和Aggrecan的AFM探针尖端。 随后分析 力距离曲线的粘附区域将产生力 相互作用。 申请人建议提案的结果 实验将明确回答IX类型的问题 胶原蛋白能够参与静电相互作用 用足够强大的Aggrecan将软骨放在下面 生理条件。