title: Analysis of limited proteolytic activity of calpain-7 using non-physiological substrates in mammalian cells.
abstract: Calpain-7 is a mammalian ortholog of a fungal non-classical calpain named PalB, which is an intracellular cysteine protease and functions in fungal alkaline adaptation in association with the endosomal sorting complex required for transport (ESCRT) system. Despite our previous finding [Osako Y et al. (2010) FEBS J 277, 4412-4426] of autolytic activity, neither physiological nor non-physiological substrates of calpain-7 have yet been identified, and experimentally useful substrates that show robust evidence of intermolecular proteolytic activity of calpain-7 are required. In this study, we found limited proteolysis of C-terminally truncated ALG-2-interacting protein X (ALIX; (ALIXΔC), but not full-length ALIX, when the mutant was co-over-expressed with calpain-7 in HEK293T cells and analyzed by western blotting. The extent of ALIXΔC cleavage by calpain-7 was enhanced by co-expression with several ESCRT proteins. We investigated whether fusion of casein, a commonly used substrate for a variety of proteases including calpains, to the Bro1 domain confers the ability to serve as a substrate of calpain-7, but no specific cleavage was observed. However, when domain 1 of calpastatin, an endogenous inhibitory protein of ubiquitous classical calpains, was fused with the Bro1 domain, the fusion protein was cleaved at the C-terminal border of subdomain B (an inhibitory center for calpains) of calpastatin. These results demonstrate for the first time that calpain-7 has limited proteolytic activity and substrate preference. Moreover, the proteolytic assay system developed enabled us to perform mutational analysis of calpain-7, which revealed the importance of not only the N-terminal microtubule-interacting and trafficking (MIT) domains but also the C-terminal C2 domain-like domains for proteolytic activity.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Maemoto
authors.forename: Satomi
authors.initials: S
authors.lastname: Kiso
authors.forename: Hideki
authors.initials: H
authors.lastname: Shibata
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Maki
mesh_terms: D000595:Amino Acid Sequence; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D002154:Calpain; C117906:Capn7 protein, human