A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively

摘要: Calpains (CAPN) are a family of Ca2+-dependent cysteine proteases that regulate various cellular functions by cleaving diverse substrates. Of the 15 mammalian calpains, CAPN8 and CAPN9 two expressed predominantly in gastrointestinal tract, where they interact to form protease complex, termed G-calpain. However, because native G-calpain exhibits highly restricted expression pattern, it has never been purified, interactions between have not characterized. Here, we clarified molecular nature using recombinant proteins transgenic mice expressing FLAG-tagged (CAPN8-FLAG). Recombinant mouse co-expressed eukaryotic systems exhibited same mobility as Blue Native-PAGE gels, CAPN8-FLAG immunoprecipitation from stomach homogenates showed was only protein associated with CAPN8-FLAG. These results indicated is heterodimer CAPN9. In addition, active purified an vitro translation system, enzymatic properties were comparable calpain-2. We found active-site mutant CAPN8, but CAPN9, compromised G-calpain's substrate cleavage activity, N-terminal helix region C-terminal EF-hands involved CAPN8/9 dimerization. Furthermore, Capn9−/− almost completely lost, whereas partially lost Capn8−/− mice. Collectively, these demonstrated function catalytic chaperone-like subunits, respectively,