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

要求提供资金，以支持正在进行的马关节疾病研究项目，该项目成果丰硕。骨关节炎是最常见的马匹关节疾病，是加拿大运动马和老年马的主要经济和福利问题。骨性关节炎的特征是关节软骨的破坏、骨的重塑和导致疼痛和关节功能丧失的炎症。其结果是退出体育活动，如果情况严重，还会实施安乐死。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，将软骨结合在一起，并赋予其抗拉强度。II型胶原分子由三条相同的a链组成，它们结合在一起形成一个三螺旋结构。这些分子相互交联，形成纤维状胶原蛋白网络的微纤维。一直以来，人们都认为关节软骨的破坏是通过软骨细胞对基质的消化来实现的。软骨的II型胶原纤维网络的破坏是骨性关节炎的一个关键的不可逆转的事件，显然与软骨的丢失有关。除了MMP1、8、13和14等胶原酶外，人们认为完整的II型胶原分子的三螺旋结构域可以抵抗大多数酶的降解。MMP13被认为是关节软骨破坏的关键因素，也是治疗骨关节炎的药物靶点。胶原酶降解II型胶原后释放的特定分子片段的检测，导致了生物标记物分析的发展，以测量体外和体内的OA疾病活动性。*我们最近报道，组织蛋白酶K(CatK)也能够裂解II型胶原的完整三螺旋，该酶在马骨性关节炎软骨中的活性高度上调。基于这些观察，我们认为在关节软骨的酶降解过程中，catK与MMP13一样重要，而且可能比MMP13更重要。我们鉴定了独特的马CatK II型胶原裂解新表位，包括C2K77(专利申请)，并提出了针对C2K77的多克隆抗体，可以在组织中检测早期马骨关节炎。*在继续这项研究计划时，我建议研究影响CatK介导的这些C2K77从马软骨中产生和释放的条件。这些释放到体液中的新表位片段的结构将被阐明，以进一步了解CatK在OA中的重要性，并开发新的免疫分析方法来检测早期马骨性关节炎。*该计划的具体目标是：1)开发一种针对CatK产生的II型胶原特异性新表位C2K77的ELISA免疫分析方法，用于定量测定CatK的活性。2)确定体外上调马关节软骨CatK活性的化学因素。3)通过对马II型胶原CatK消化产生的C2K77的所有片段的序列结构特征分析，确定OA中产生的优势片段(S)，并建立夹心免疫分析方法。4)评价基于C2K77的马生物标志物检测体液中OA的新方法。这项在马骨性关节炎研究的一个新的新兴领域的研究计划有望促进对CatK参与马骨性骨性关节炎的II型胶原降解和软骨破坏的了解。开发一种基于体液分析的敏感、特异的早期马骨性关节炎诊断试验(S)对马业极其重要，因为骨性关节炎是一个重大的福利和经济问题。这项研究的结果也有可能对人类和狗在骨性关节炎方面的研究产生影响。

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.