BBA - Proteins and Proteomics 1868 (2020) 140411 Contents lists available at ScienceDirect BBA - Proteins and Proteomics journal homepage: www.elsevier.com/locate/bbapap A muscle-specific calpain, CAPN3, forms a homotrimer T ⁎ ⁎ Shoji Hata , Naoko Doi, Fumiko Shinkai-Ouchi, Yasuko Ono ,1 From Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science (TMiMS), 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan ARTICLE INFO ABSTRACT Keywords: Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations CAPN3 in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles Calpain in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal Protease penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To Dimer analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide Trimer gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant EF-hand Skeletal muscle CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains. 1. Introduction catalytic subunits consist of an N-terminal anchor helix peptide fol- lowed by a highly conserved protease (CysPc) domain consisting of Calpains (Clan CA-C2, EC3.4.22.17) constitute a family of in- protease core subdomains PC1 and PC2, a calpain-type β-sandwich tracellular Ca2+-requiring cysteine proteases present in a wide range of (CBSW) domain, and a penta-EF-hand (PEF) domain. CAPNS1 contains species from bacteria to humans [1–3]. Mammals have 15 calpain genes a glycine-rich (GR) domain and a PEF domain, and functions as a mo- (CAPN1–3 and 5–16) with ubiquitous or tissue-specific expression. lecular chaperone for CAPN1 and CAPN2. Contrary to the definition of Calpains regulate multiple biological phenomena, including apoptosis, the EF-hand as Ca2+-binding motif, the fifth EF-hand (EF5) of PEF cell cycle, myoblast fusion, and membrane repair, depending on the domains of both subunits does not function as Ca2+-binding site, but functions of their substrates [1]. Accordingly, aberrant calpain activity instead contributes to the interface between the two subunits. In ad- caused by cellular abnormalities or gene mutations is associated with dition, the Ca2+ requirement of calpain is governed by its CysPc do- many pathological situations, including developmental failure, neuro- main. In the absence of Ca2+, calpain-1 and calpain-2 are catalytically degenerative disease, muscular dystrophies, and ophthalmic disorders inactive because PC1 and PC2 are far apart, preventing formation of the [4–14]. The diverse outcomes of calpain activity are the consequence of active site. The binding of Ca2+ to PC1 and PC2 induces considerable limited cleavage of specific substrates, although the underlying mole- structural changes required for an active protease core formation cular mechanisms remain unclear. Hence, calpain is attracting attention [2,15–19]. as a promising target for the treatment of these diseases [3]. CAPN3, the first identified tissue-specific calpain, is predominantly Of the mammalian calpains, the abundant, ubiquitously expressed expressed in skeletal muscle [20]. Several years after its discovery, the calpain-1 and -2 are the best characterized. They exist as heterodimers CAPN3 gene was identified as responsible for limb girdle muscular of a distinct 80-kDa catalytic subunit, CAPN1 for calpain-1 and CAPN2 dystrophy type 2A (LGMD2A) [5]. Studies using mice with genetic for calpain-2, and a common 28-kDa regulatory subunit, CAPNS1. The deficiencies in the Capn3 gene showed that CAPN3 plays critical roles as Abbreviations: CysPc, calpain-type Cys protease conserved domain; PC, protease core domain; PEF, penta-EF-hand domain; NS, N-terminal sequence; IS, internal sequence; BN-PAGE, Blue-Native PAGE; FL, full-length; Ab, antibody; GA, Glutaraldehyde; IP, immunoprecipitation; iMOC, intermolecular complementation; KO, knock-out; KI, knock-in; aa, amino acid residues. ⁎ Corresponding author. E-mail addresses: [email protected] (S. Hata), [email protected] (Y. Ono). 1 Lead contact. https://doi.org/10.1016/j.bbapap.2020.140411 Received 16 January 2020; Received in revised form 4 March 2020; Accepted 11 March 2020 Available online 19 March 2020 1570-9639/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). S. Hata, et al. BBA - Proteins and Proteomics 1868 (2020) 140411 a sensor of muscle stress under various conditions, e.g., adaptive muscle Chemiluminescence signals were detected by the ImageQuant LAS 4000 growth and remodeling after atrophy and exercise [21–25]. Moreover, imaging system (GE Healthcare). Images were processed for presenta- CAPN3 functions as a component of the Ca2+ transfer system in muscle tion using Adobe Photoshop CC (Adobe Systems). cells, although the molecular mechanisms remain elusive [26,27]. At the level of primary structure, CAPN3 has a similar domain structure to those of CAPN1 and CAPN2, except for three additional 2.4. Protein expression in COS7 cells sequences at the N-terminus (NS, 47 amino acid residues [aa]), within fi the CysPc domain (IS1, 48 aa), and between the CBSW and PEF do- COS7 cells were cultured in Dulbecco's modi ed Eagle's medium μ mains (IS2, 77 aa) (Fig. 3A). Several unique enzymatic and structural supplemented with 10% heat-inactivated fetal bovine serum. Total 3 g 5 properties of CAPN3 have been identified [28–32]. Some are due to the of the expression vectors were transfected into 3 × 10 COS7 cells μ presence of the additional sequences, whereas others are not. Based on using 12 l of PEI MAX 40 K (Polysciences). The cells were harvested μ ff crystallization of the CAPN3 PEF domain homodimer [32] and a gel 24 h after transfection and lysed by sonication in 150 l of lysis bu er filtration analysis of the catalytically inactive CAPN3:Cys129Ser mutant [10 mM Tris/HCl (pH 7.5), 1 mM EDTA/CsOH, 1 mM DTT, protease [33], it has been proposed that CAPN3 homodimerizes by pairing of the inhibitor cocktail (Roche)]. After centrifugation at 20,600 ×g for EF5 domains. This is compatible with the idea that PEF-containing 15 min, the soluble fraction was recovered and subjected to SDS-PAGE proteins tend to form either homodimers or heterodimers [34]. To date, or BN-PAGE, followed by western blotting. however, there is no definitive evidence regarding the oligomeric state of CAPN3. 2.5. Experimental animals Inspired by our success in analyzing G-calpain, a heterodimer of CAPN8 and CAPN9 [35], we explored the potential of blue native PAGE All procedures using experimental animals were approved by the (BN-PAGE) for characterizing the molecular composition of CAPN3 Animal Use and Care Committee of the Tokyo Metropolitan Institute of prepared from skeletal muscle extracts, as well as recombinant CAPN3 Medical Science. Housing of the mice and acquisition of tissue were expressed in cultured cells. To our surprise, CAPN3 from both pre- performed in the Tokyo Metropolitan Institute of Medical Science ac- parations was detected as a complex with a molecular weight > cording to the committee's guidelines. Generation and phenotypic 240 kDa. Further investigation revealed that CAPN3 forms a homo- characterization of CAPN3:C129S KI (Capn3C129S/C129S) mice, which trimer. Together, our results show that the molecular entity of active express CAPN3:C129S, a protease-inactive mutant, were described − − CAPN3 complex is more dynamic than what would be expected for a previously [23,27]. Capn3 KO (Capn3 / ) mice were generated by calpain. replacing exon 3, which contains the codon for the active-site residue Cys, with a neomycin-resistance gene cassette as previously described 2. Materials and methods [10]. Female mice, aged 25 weeks, were used. Mice were sacrificed by cervical dislocation under anesthesia using isoflurane (DS Pharma An- 2.1. Antibodies imal Health Inc.). Tibialis anterior muscles were excised, and frozen in liquid nitrogen. Forty muscle sections (20 μm-thickness) sliced by The following antibodies were obtained from the indicated sources cryostat (Leica) were homogenized in lysis buffer. The soluble fraction – and suppliers: anti CAPN3-IS2 goat polyclonal antibody [36], an- was recovered after centrifugation at 20,600 ×g for 15 min, and sub- – ti CAPN3-NS rabbit polyclonal antibody (RP4-Calpain3, Triple Point jected to SDS-PAGE and BN-PAGE (10 μg per lane). Biologics), anti-CAPN9 goat polyclonal antibody (V-18, Santa Cruz Biotechnology), anti-FLAG mouse monoclonal antibody (clone M2, Sigma-Aldrich), anti-MYC mouse monoclonal antibody (clone 4A6, 2.6. Immunoprecipitation Merck Millipore), anti-HA mouse monoclonal antibody (clone 6E2, Cell Signaling Technology), HRP-conjugated anti–mouse or rabbit IgG Total 3 μg of the expression vectors