A PROPOSAL FOR CONTINUATION OF A RESEARCH PROGRAM TO INVESTIGATE THE ROLE OF CATHEPSIN K IN EQUINE OSTEOARTHRITIS (OA) AND CREATE NEW DIAGNOSTIC ASSAYS FOR THE EARLY DETECTION OF OA

资金要求支持一个正在进行的和高生产力的研究计划，在马关节疾病。骨关节炎（OA），最常见的马关节疾病，是一个主要的经济和福利问题，在运动和老年加拿大马。OA的特征在于关节软骨的破坏、骨重塑和炎症，其导致疼痛和关节功能丧失。其结果是从体育活动中退休，如果严重的话，安乐死。关节软骨的细胞外基质由常驻软骨细胞维持。最丰富和机械上必需的结构软骨基质分子是II型胶原。它形成了一个广泛的网络，将软骨保持在一起，并赋予其拉伸强度。II型胶原蛋白分子由三条相同的α链组成，它们联合收割机形成三股螺旋。这些分子交联形成纤维状胶原网络的微纤维。长期以来，人们已经认识到OA中关节软骨的破坏是由软骨细胞蛋白酶消化基质介导的。软骨的II型胶原纤维网络的破坏是OA中的关键不可逆事件，并且与软骨损失明显相关。据信，完整的II型胶原分子的三螺旋结构域抵抗除胶原酶如MMP 1、8、13和14之外的大多数蛋白酶的降解。MMP 13被认为在关节软骨破坏中具有关键重要性，并且是OA的药物靶点。通过胶原酶降解II型胶原后释放的特异性分子片段的检测，导致了生物标志物测定的发展，以测量体外和体内OA疾病活性。我们最近报道，组织蛋白酶K（catK）也能够切割完整的三螺旋的II型胶原蛋白，这种酶的活性在马OA软骨中高度上调。基于这些观察，我们认为catK在关节软骨的酶促降解中与MMP 13一样重要，并且可能更重要。我们鉴定了独特的马catK II型胶原特异性切割新表位，包括C2K77（已提交专利），并提出了C2K77的多克隆抗体，其可以检测组织中的早期马OA。在继续这项研究计划，我建议研究的条件，影响catK介导的产生和释放这些C2K77从马软骨。这些新表位的片段释放到体液中的结构将被阐明，以进一步了解catK在OA中的重要性，并开发新的免疫测定来检测早期马OA。该计划的具体目标是：1）开发针对catK产生的II型胶原特异性新表位C2K77的ELISA免疫测定，用于定量catK活性。2)鉴定在体外马关节软骨中上调catK活性的化学因子。3)通过表征所有片段的序列结构，确定OA中产生的优势片段，这些片段含有通过catK消化马II型胶原蛋白在体内产生的C2K77，并开发夹心免疫测定法。4)评估基于C2K77的新型马生物标志物检测方法，用于检测体液中的OA。在马OA研究的一个新的新兴领域的调查程序持有的承诺，以推进知识的catK参与II型胶原降解和马OA软骨破坏。由于OA是一个主要的福利和经济问题，因此基于体液分析开发用于早期马OA的敏感、特异性诊断测试对马产业极其重要。这项研究的结果也有可能影响人类和犬类的OA研究。

Funds are requested for support of an ongoing and highly productive research program in equine joint disease. Osteoarthritis (OA), the most common equine joint disease, is a major economic and welfare problem in athletic and aged Canadian horses. OA is characterized by destruction of the articular cartilage, bone remodeling and inflammation that causes pain and loss of joint function. The outcome is retirement from athletic activity and, if severe, euthanasia. The extracellular matrix of articular cartilage is maintained by resident chondrocytes. The most abundant and mechanically essential structural cartilage matrix molecule is type II collagen. It forms an extensive network that holds cartilage together and gives it its tensile strength. Type II collagen molecules consist of three identical a chains that combine to form a triple helix. These molecules are cross-linked to form the microfibrils of the fibrillar collagen network. It has long been recognized that the destruction articular cartilage in OA is mediated by chondrocyte protease digestion of the matrix. Destruction of the type II collagen fibrillar network of cartilage is a key irreversible event in OA and clearly linked to cartilage loss. It was believed that the triple helical domain of intact type II collagen molecules is resistant to degradation by most proteases except collagenases such as MMP1, 8, 13 and 14. MMP 13 is considered of key importance in articular cartilage breakdown and a drug target for OA. Detection of specific molecular fragments, released following degradation of type II collagen by collagenases, has resulted in the development of biomarker assays to measure OA disease activity in vitro and in vivo. We recently reported that cathepsin K (catK) is also capable of cleavage of the intact triple helix of type II collagen, and activity of this enzyme is highly upregulated in equine OA cartilage. Based on these observations, we believe that catK is as important, and potentially more important, than MMP 13 in the enzymatic degradation of articular cartilage. We identified unique equine catK type II collagen specific cleavage neoepitopes, including C2K77 (patent filed), and have raised polyclonal antibodies to C2K77 that can detect early equine OA in tissues. In continuation of this research program, I propose to study the conditions that influence catK-mediated generation and release of these C2K77 from equine cartilage. The structures of these neoepitope-containing fragments released into body fluids will be elucidated to further understand the importance of catK in OA and to develop new immunoassays to detect early equine OA. Specific objectives of the program are to: 1) Develop an ELISA immunoassay directed at a catK-generated type II collagen specific neoepitope C2K77 for quantitation of catK activity. 2) Identify chemical factors that upregulate catK activity in equine articular cartilage in vitro. 3) Establish the dominant fragment(s) generated in OA by characterization of sequence structures of all fragments containing the C2K77 produced in vivo by catK digestion of equine type II collagen and develop sandwich immunoassays. 4) Assess the new equine biomarker assays based on C2K77 for the detection of OA in body fluids. This program of investigation in a novel emerging field in equine OA research holds the promise to advance knowledge of catK involvement in type II collagen degradation and destruction of cartilage in equine OA. The development of a sensitive, specific diagnostic test(s) for early equine OA based on analysis of body fluids would be extremely important for the equine industry as OA is a major welfare and economic issue. The results of this research also have the potential to impact both human and canine research in OA.