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

关于继续开展一项研究计划的提案，以调查组织蛋白酶 K 在马骨关节炎 (OA) 中的作用并为 OA 的早期检测创建新的诊断方法

• 批准号: RGPIN-2014-03836
• 负责人: Laverty, Sheila
• 金额: $ 3.86万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656790
• 关键词: PROPOSAL; CONTINUATION; RESEARCH; PROGRAM; INVESTIGATE

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.

需要资金支持正在进行的、高效的马关节疾病研究项目。骨关节炎 (OA) 是最常见的马关节疾病，是加拿大运动马和老年马的主要经济和福利问题。 OA 的特点是关节软骨破坏、骨重塑和炎症，导致疼痛和关节功能丧失。结果是退出体育活动，如果严重的话，将被安乐死。*关节软骨的细胞外基质由常驻软骨细胞维持。最丰富且机械必需的结构软骨基质分子是 II 型胶原蛋白。它形成一个广泛的网络，将软骨固定在一起并赋予其抗拉强度。 II 型胶原蛋白分子由三个相同的 a 链组成，这些 a 链结合形成三螺旋。这些分子交联形成纤维状胶原蛋白网络的微纤维。人们早就认识到，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 II 型胶原纤维网络的破坏是 OA 中的一个关键的不可逆事件，并且与软骨损失明显相关。据信，完整 II 型胶原分子的三螺旋结构域可抵抗除胶原酶（如 MMP1、8、13 和 14）以外的大多数蛋白酶的降解。MMP 13 被认为在关节软骨破坏中至关重要，也是 OA 的药物靶点。对胶原酶降解 II 型胶原后释放的特定分子片段的检测导致了生物标志物测定法的发展，以测量体外和体内 OA 疾病活动。 *我们最近报道，组织蛋白酶 K (catK) 也能够裂解 II 型胶原蛋白的完整三螺旋，并且该酶的活性在马 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，甚至可能更重要。我们鉴定了独特的马 catK II 型胶原蛋白特异性裂解新表位，包括 C2K77（已申请专利），并制备了针对 C2K77 的多克隆抗体，可以检测组织中的早期马 OA。*在该研究计划的继续中，我建议研究以下条件：影响 catK 介导的马软骨中这些 C2K77 的生成和释放。这些释放到体液中的含有新表位的片段的结构将被阐明，以进一步了解 catK 在 OA 中的重要性，并开发新的免疫测定法来检测早期马 OA。 *该计划的具体目标是： 1) 开发一种针对 catK 生成的 II 型胶原蛋白特异性新表位 C2K77 的 ELISA 免疫测定法，用于定量 catK 活性。 2) 鉴定体外上调马关节软骨中 catK 活性的化学因子。 3) 通过对马 II 型胶原的 catK 消化体内产生的含有 C2K77 的所有片段的序列结构进行表征，确定 OA 中产生的主要片段，并开发夹心免疫测定法。 4) 评估基于 C2K77 的新马生物标志物检测方法，用于检测体液中的 OA。这项针对马 OA 研究新兴领域的研究计划有望增进对 catK 参与马 OA 中 II 型胶原降解和软骨破坏的了解。基于体液分析的早期马 OA 的敏感、特异诊断测试的开发对于马业来说极其重要，因为 OA 是一个重大的福利和经济问题。这项研究的结果也有可能影响人类和犬类的 OA 研究。