INTERMOLECULAR PHYSICAL INTERACTIONS IN CARTILAGE

这项研究工作的长期目标是找到预防性的 和/或骨关节炎的治疗措施 这种疾病的分子和亚分子发病机制。 这一建议的核心假设是， IX型胶原蛋白上亚分子局部电荷域的大小 在胶原纤维和聚集蛋白聚糖之间的相互作用中是重要的 软骨细胞外基质（ECM）中的分子。 II型 假设胶原纤维连接到聚集的蛋白聚糖 通过带负电荷的 聚集蛋白聚糖糖胺聚糖侧链上的硫酸酯基团， 理论上高度带正电荷的非胶原蛋白4（NC 4）结构域 IX型胶原，其共价结合至IX型胶原的表面， II纤维。 有大量的间接证据表明， 相互作用在获得和保持正常粘弹性中是至关重要的 ECM的生物力学功能。 拟议的工作将提供一个 直接测量这种相互作用。 此外，体外亚- 构象和电荷分布的分子尺度测量 II型和IX型胶原蛋白和聚集蛋白聚糖的纯化单体将 获得。 这一信息将是至关重要的发展， 下一代基于分子的ECM理论模型 生物力学 该提案的具体目标是：1）形象 单个表面驻留II型和IX型胶原和聚集蛋白聚糖 分子在环境和体外条件下; 2）测量力 每个分子产生的场; 3）测量 硫酸软骨素（CS）与II型和IX型之间相互作用 胶原蛋白;和4）测量聚集蛋白聚糖和胶原蛋白之间的相互作用力。 II型和IX型胶原分子。 这些目标将会实现 使用原子力显微镜来探测固定在 面 通过分析标准卡扣接触部分的数据， 力-距离曲线、胶原蛋白上电荷的大小以及 将获得聚集蛋白聚糖分子（Aim 2）。末端的小尺寸 的AFM探针针尖允许这种测量与子- 分子空间分辨率目的3和4涉及共价连接 的CS和聚集蛋白聚糖到AFM探针尖端。 后续分析 力-距离曲线的粘合区域将产生 互动 申请人建议， 实验将明确地回答IX型是否 胶原能够参与静电相互作用 聚集蛋白聚糖的强度足以将软骨结合在一起， 生理条件。

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.