COLLAGEN CROSS-LINKING IN SKELETAL AGING AND DISEASE

描述(由申请人提供)：目标是在蛋白质水平上精确详细地定义主要骨骼组织胶原蛋白赖氨酰氧化酶介导的交联机制是如何不同的。研究遗传性疾病的见解和问题表明，我们还没有很好地了解胶原蛋白交联化学的局部特征及其潜在的控制机制，不足以将形式与功能联系起来。同样清楚的是，胶原蛋白的翻译后质量会随着年龄和疾病的变化而变化，这种变化会影响酶调节的交联物的基本模式。越来越多的证据表明，个体之间的这种差异是骨质疏松症、骨关节炎和其他退行性骨骼疾病的风险因素。该实验计划使用先进的质谱学方法和针对交联域的定制抗体来研究胶原交联物的生物化学。同时，从组织研究中获得的见解和分子工具将被应用于推进现有基于胶原交联物的生物标记物分析的知识，并开发新的非侵入性监测骨骼和关节疾病的生物标记物。 其具体目的包括：1)明确骨胶原的详细翻译后化学，包括吡啶啉、吡咯和酮亚胺交联链的位置和化学形式的使用，以及在骨质疏松症、成骨不全和其他代谢改变条件下随年龄而出现的差异；2)类似详细地比较软骨、肌腱、韧带和关节囊的非矿化骨胶原的交联性；3)确定III型胶原作为软骨和其他非矿化性骨骼组织细胞外基质的调节剂的交联性；4)根据组织研究中对胶原交联机制的认识，改进并开发临床有用的生物标记物。研究设计和方法的重点是应用蛋白质分析中的质谱学和其他先进方法来确定组织胶原结构的细节，然后使用细胞培养来探索组织特异性交联物的多样性以及在衰老和疾病中发生的变化的候选调控机制。

DESCRIPTION (provided by applicant): The goal is to define in precise detail at the protein level how the lysyl oxidase-mediated cross-linking mechanisms of the major skeletal tissue collagens differ. Insights and questions from studying heritable disorders make it clear that we do not yet understand the local features of collagen cross-linking chemistry and their underlying control mechanisms well enough to relate form to function. It is also clear that the posttranslational quality of collagen can change with age and disease in ways that affect the basic pattern of enzyme-regulated cross-linking. There is growing evidence that such differences between individuals are a risk factor for osteoporosis, osteoarthritis and other degenerative skeletal disorders. The experimental plan targets the biochemistry of collagen cross-linking using advanced mass spectroscopic methods and custom antibodies against cross-linking domains. In parallel, the insights and molecular tools gained from tissue studies will be applied to advance knowledge on existing biomarker assays based on collagen cross-links and develop new biomarkers for monitoring bone and joint disorders non-invasively. The specific aims include: 1) defining the detailed post-translational chemistry of bone collagen including chain usage in the placement and chemical forms of pyridinoline, pyrrole and keto-imine cross-links and differences that occur with age in osteoporosis, osteogenesis imperfecta and other conditions of altered metabolism; 2) compare in similar detail the cross-linking properties of non-mineralized skeletal collagens of cartilages, tendon, ligament and joint capsule; 3) determine the cross-linking properties of type III collagen as a modifier of the extracellular matrix of cartilage and other non-mineralized skeletal tissues; 4) improving and, developing clinically useful biomarkers from the insights gained on collagen cross-linking mechanisms from the tissue studies. The emphasis in the research design and methods is to apply mass spectrometry and other advanced methods in protein analysis to determine details of tissue collagen structure, then use cell culture to explore candidate control mechanisms responsible for the diversity in tissue-specific cross-linking and the changes that occur in aging and disease.