RRC ID 48551
著者 Hata S, Kitamura F, Yamaguchi M, Shitara H, Murakami M, Sorimachi H.
タイトル A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively.
ジャーナル J Biol Chem
Abstract 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 are two that are expressed predominantly in the gastrointestinal tract, where they interact to form a protease complex, termed G-calpain. However, because native G-calpain exhibits a highly restricted expression pattern, it has never been purified, and the interactions between CAPN8 and CAPN9 have not been characterized. Here, we clarified the molecular nature of G-calpain by using recombinant proteins and transgenic mice expressing FLAG-tagged CAPN8 (CAPN8-FLAG). Recombinant mouse CAPN8 and CAPN9 co-expressed in eukaryotic expression systems exhibited the same mobility as native mouse G-calpain in Blue Native-PAGE gels, and CAPN8-FLAG immunoprecipitation from stomach homogenates of the transgenic mice showed that CAPN9 was the only protein that associated with CAPN8-FLAG. These results indicated that G-calpain is a heterodimer of CAPN8 and CAPN9. In addition, active recombinant G-calpain was expressed and purified using an in vitro translation system, and the purified protease exhibited enzymatic properties that were comparable with that of calpain-2. We found that an active-site mutant of CAPN8, but not CAPN9, compromised G-calpain's substrate cleavage activity, and that the N-terminal helix region of CAPN8 and the C-terminal EF-hands of CAPN8 and CAPN9 were involved in CAPN8/9 dimerization. Furthermore, CAPN8 protein in Capn9-/- mice was almost completely lost, whereas CAPN9 was only partially lost in Capn8-/- mice. Collectively, these results demonstrated that CAPN8 and CAPN9 function as catalytic and chaperone-like subunits, respectively, in G-calpain.
巻・号 291(53)
ページ 27313-27322
公開日 2016-12-30
DOI 10.1074/jbc.M116.763912
PII S0021-9258(20)34400-8
PMID 27881674
PMC PMC5207157
MeSH Amino Acid Sequence Animals Calpain / metabolism* Catalysis Cells, Cultured Female Gastric Mucosa / metabolism* Mice Mice, Inbred C57BL Mice, Transgenic Protein Isoforms Recombinant Proteins / metabolism* Sequence Homology, Amino Acid
IF 4.238
引用数 5
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
遺伝子材料 pCALNL5 (RDB01862)