Santamaria Salvatore, Yamamoto Kazuhiro
Centre for Haematology, Imperial College London, London, UK.
Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.
Methods Mol Biol. 2020;2043:125-136. doi: 10.1007/978-1-4939-9698-8_11.
Aggrecan is a major matrix component of articular cartilage, and its dysregulated proteolysis is a crucial event in the pathogenesis of arthritis. Aggrecanases, members of ADAMTS family, play a pivotal role in aggrecan degradation with ADAMTS-4 and ADAMTS-5 being key enzymes. Cleavage events mediated by ADAMTSs are highly specific and very well characterized; therefore, it is possible to investigate aggrecanolysis by using antibodies reacting with the new N- and C-termini of the cleavage products (neoepitope antibodies). Here, we present a method for analyzing dynamic aggrecanolysis by Western blotting using neoepitope antibodies in combination with antibodies against total aggrecan fragments. The protocol is robust and has a broad application for investigation of aggrecanase activity in vitro and ex vivo.
聚集蛋白聚糖是关节软骨的主要基质成分，其蛋白水解失调是关节炎发病机制中的关键事件。ADAMTS家族成员聚集蛋白聚糖酶在聚集蛋白聚糖降解中起关键作用，其中ADAMTS-4和ADAMTS-5是关键酶。ADAMTS介导的切割事件具有高度特异性且特征明确；因此，使用与切割产物的新N端和C端反应的抗体（新表位抗体）来研究聚集蛋白聚糖分解是可行的。在此，我们介绍一种通过蛋白质印迹法，结合使用新表位抗体和针对总聚集蛋白聚糖片段的抗体来分析动态聚集蛋白聚糖分解的方法。该方案稳健，在体外和体内研究聚集蛋白聚糖酶活性方面具有广泛应用。
