BBA - and Proteomics 1868 (2020) 140411

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BBA - Proteins and Proteomics

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A muscle-specific , 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 family, is abundant in skeletal muscle. Mutations CAPN3 in the CAPN3 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 (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 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 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. 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 were transfected into 3 × 105 (Dako), and HRP-conjugated anti–goat IgG (Nichirei). COS7 cells using 12 μl of PEI MAX 40 K. The cells were harvested 24 h after transfection, and lysed with 500 μlofIPbuffer [20 mM Tris/HCl 2.2. Generation of expression vectors (pH 7.5), 4 mM EDTA/CsOH, 150 mM CsCl, 0.5%(v/v) Nonidet P-40, protease inhibitor cocktail (Roche)]. After centrifugation at 20,600 ×g cDNAs encoding full-length and truncated forms of human CAPN3 for 15 min, the soluble fractions were recovered, and incubated with 10 and full-length human sorcin were amplified by PCR using Phusion µl of anti-FLAG magnetic beads (Sigma-Aldrich) or anti-FLAG M2 affi- High Fidelity DNA polymerase (Thermo Fisher Scientific). The ampli- nity gel (Sigma-Aldrich) equilibrated with IP buffer for 1 h at 4 °C. After fied cDNAs were cloned into modified pcDNA3.1 vectors to produce the incubation, the beads or gel were washed four times with IP buffer, proteins with an N-terminal FLAG, HA, or MYC-tag or into a modified and the bound proteins were eluted with 3 × FLAG peptides (Protein pSRD vector [31]. The CAPN3 point mutants were generated by PCR- Ark). The eluate was mixed with SDS-PAGE sample buffer, and ana- mediated site-directed mutagenesis as described previously [37]. Ex- lyzed by SDS-PAGE and western blotting. pression vectors for mouse CAPN3 splicing variants (CAPN3ΔIS, Fig. For sequential immunoprecipitation, 10 μg each of the expression S1B) were constructed as previously described [38]. The sequences of vectors (total 30 μg) were co-transfected into 4 × 106 COS7 cells using the constructed vectors were verified by DNA sequencing. 120 μl of PEI MAX 40 K. The cells harvested 24 h after transfection were lysed with 1.5 ml of IP buffer. The soluble fractions recovered after 2.3. SDS-PAGE, BN-PAGE, and western blotting centrifugation at 20,600 ×g for 15 min were incubated with 50 μlof anti-FLAG M2 magnetic beads equilibrated with IP buffer for 1 h at 4 °C. Protein samples were separated on 7.5–15% gradient precast gels After washing the beads four times with IP buffer, the bound proteins (Nacalai Tesque) for SDS-PAGE and 4–16% Native PAGE Bis-Tris gra- were eluted with 3 × FLAG peptides, and the eluate was diluted in IP dient gels (Thermo Fisher Scientific) for BN-PAGE. After electrophor- buffer and incubated for 30 min at 4 °C with 50 μl of Dynabeads Protein esis, the samples were transferred onto PVDF membranes (Millipore). G (Invitrogen) conjugated with 1.5 μg of anti-MYC antibody. After The membranes were probed with primary and HRP-conjugated sec- washing the beads with IP buffer, the final immunocomplex was eluted ondary antibodies, and then visualized using the POD immunostaining with 0.2 M glycine (pH 2.7). The eluate was immediately neutralized kit (Wako), Clarity Western ECL Substrate (Bio-Rad), or SuperSignal with Tris-HCl (pH 9.5), mixed with SDS-PAGE sample buffer, and West Dura Extended Duration Substrate (Thermo Fisher Scientific). analyzed by SDS-PAGE and western blotting.

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2.7. Density gradient centrifugation (Fig. 2B). CAPN3:C129S proteins N-terminally fused with FLAG-, MYC-, or HA-tag were co-expressed in COS7 cells in the indicated combina- This analysis was performed as previously described with some tions (top list), and the cell lysate was subjected to sequential im- modifications [39,40]. A linear gradient of 10–50% OptiPrep (Alere munoprecipitation using anti-FLAG and anti-MYC antibodies. In the Technologies AS) in sedimentation buffer [50 mM Tris/Cl (pH 7.5), final precipitant, HA-CAPN3:C129S was detected only in sample 3 1 mM EDTA/CsOH, 100 mM CsCl] was made by allowing a dis- (Fig. 2B, lane 3 in right panel). Taken together, these results indicated continuous gradient to diffuse. FLAG-CAPN3:C129S complex eluted that CAPN3 forms a homotrimer. with 3 × FLAG peptide by immunoprecipitation was layered on top of the gradient and centrifuged at 40,000 rpm for 30 h at 4 °C in a 3.2. PEF domain is necessary for CAPN3 trimerization, but forms a Beckman-Coulter SW 55 Ti rotor. Fractions (0.12 ml per fraction) were homodimer by itself collected from top to bottom, and analyzed by SDS-PAGE and western blotting. A protein marker, catalase (~240 kDa) (Tokyo Chemical In- To identify the molecular region responsible for CAPN3 trimeriza- dustry), was sedimented under the same condition. Density was esti- tion, we examined the trimerization activity of a series of deletion mated from the A300 of each fraction to calibrate the gradient. mutants of CAPN3 fused with an N-terminal FLAG-tag (Fig. 3A). To exclude autolytic effects, the C129S mutation was introduced where 2.8. Cross-linking appropriate. Each mutant was expressed in COS7 cells and subjected to GA crosslinking, followed by SDS-PAGE and western blotting (Fig. 3B). Cross-linking was performed as previously described [41] with some Intriguingly, ΔPEF did not exhibit a mobility shift corresponding to modifications. The harvested COS7 transfectants were lysed by soni- trimerization (lane 5 vs. 6); instead, crosslinked IS2-PEF and PEF were cation in lysis buffer, and the soluble fractions were recovered as de- detected at a position that could be interpreted as a homodimer (lane scribed in Section 2.4. Ten μg of the soluble fractions were incubated 17 vs. 18, and 19 vs. 20, respectively). ΔEF5 (lane 3 vs. 4), ΔNS-PC1 with or without a final concentration of 0.05% GA for 10 min at room (lane 9 vs. 10), 58 K (lane 13 vs. 14), and ΔNS-PC1/2 (lane 15 vs. 16) temperature. The reactions were stopped by addition of SDS-PAGE were barely detectable as high–molecular weight smear signals when sample buffer, and then analyzed by SDS-PAGE and western blotting. crosslinked, and the corresponding signals were also observed by BN- PAGE (Fig. S1A, lanes 2 and 5–7). Therefore, we considered these 3. Results mutants to be aggregation-prone due to structural instability. By con- trast, ΔNS was still capable of forming a trimer, as suggested by de- 3.1. CAPN3 is detected as a homotrimer tection of a complex at the corresponding position (Fig. 3B, lanes 7 and 8), which was also observed by BN-PAGE (Fig. S1A, lane 4). We then CAPN3 exerts rapid and exhaustive autolytic activity in almost all investigated the involvement of the CAPN3-specific insertions, IS1 and protein expression systems examined to date [28,42,43]. Therefore, IS2, in trimerization. In BN-PAGE, CAPN3 lacking both IS1 and IS2 most of the constructs we used in this study harbor the Cys129Ser (CAPN3ΔIS, 84 kDa) was detected at a position corresponding to a (C129S) mutation, which inactivates CAPN3, thereby stabilizing the trimeric form (252 kDa) (Fig. S1B, lane 1). These results led us to expressed protein. conclude that the PEF domain is necessary but not sufficient for tri- To determine the molecular size of the CAPN3-containing complex, merization, and that NS, IS1, and IS2 are not necessary for trimeriza- and in particular to determine whether full-length CAPN3 forms a tion. homodimer, we first performed BN-PAGE analysis. We previously showed that this system is capable of detecting a ~160-kDa hetero- 3.3. The CBSW domain contributes to CAPN3 trimerization through the dimer of two gastrointestinal tract–specific calpains, CAPN8 and PEF domain CAPN9, in mouse stomach homogenate (Fig. 1B, lane 6) [35]. Using the same conditions, in parallel with native CAPN3 and CAPN3:C129S ex- To identify the additional domain(s) required for trimerization, pressed in skeletal muscles of wild-type and CAPN3:C129S knock-in each mutant shown in Fig. 3A, except for ΔNS and ΔEF5, was evaluated (KI) mice, respectively, we analyzed FLAG-tagged human as to its ability to form a heterotrimer with two molecules of full-length CAPN3:C129S expressed in COS7 cells (Fig. 1). Surprisingly, CAPN3 CAPN3. Cell lysate from COS7 cells expressing each mutant in combi- from all three preparations had the same mobility, between 242 and nation with MYC- and HA-CAPN3:C129S was subjected to sequential 480 kDa, corresponding to 3–4 times larger than the molecular weight immunoprecipitations using anti-FLAG and anti-MYC antibodies. As of the 94-kDa monomer (Fig. 1B, lanes 1–5). Density gradient analysis shown in Fig. 3C, HA-CAPN3:C129S was detected in the final im- also revealed that FLAG-CAPN3:C129S complex immunopurified with munoprecipitate from ΔNS-PC1, 58K, and ΔNS-PC1/2 transfectants, as anti-FLAG antibody from COS7 cells was enriched in fractions corre- well as cells expressing full-length CAPN3 and ΔNS (lanes 9 and 11–14 sponding to a molecular weight greater than 240 kDa (Fig. 1C). These in the lower right panel). This indicated that, in addition to the PEF observations suggested that CAPN3 forms a heterocomplex containing domain, at least the CBSW domain is required for trimerization. two CAPN3 molecules and additional component(s), a homotrimer In this experiment, the PEF and IS2-PEF mutants were faintly de- (theoretical molecular weight 282 kDa), or a tetramer (376 kDa). tected with MYC-CAPN3:C129S in the final immunoprecipitate, which We also investigated the molecular weight of CAPN3 complex by did not contain HA-CAPN3:C129S (lanes 15 and 16 in the lower right glutaraldehyde (GA)-mediated protein crosslinking. FLAG-tagged panel). These interactions were also observed weakly by im- CAPN3:C129S or sorcin, a 22-kDa PEF family protein that forms a munoprecipitation using either anti-FLAG or anti-MYC antibody from homodimer, was expressed in COS7 cells, and the cell lysates were COS7 cells expressing MYC-CAPN3:C129S in combination with IS2-PEF treated with GA prior to SDS-PAGE and western blotting. As shown in or PEF (Fig. S2A, lanes 8 and 9 in right two panels; Fig. S2B, lanes 4 and Fig. 2A, crosslinked sorcin was detected at the position corresponding 5 in right two panels). These results indicated that PEF can form a to a dimer (44 kDa; lane 2, arrowhead). Under the same condition, heterodimer with full-length CAPN3 at a trace level. BN-PAGE analysis crosslinked CAPN3 was detected between 250 and 315 kDa (lane 4, of a cell lysate co-expressing PEF and MYC- and HA- CAPN3:C129S arrowhead). This result allowed us to exclude the possibility that yielded no signals other than those corresponding to a homotrimer CAPN3 forms a homotetramer. Hence, we investigated the possibility composed of MYC- and/or HA-CAPN3:C129S even in the presence of that it forms a homotrimer. PEF (Fig. S2C, lane 1). Due to the dimeric properties of PEF and the We next performed a tandem immunoprecipitation assay to de- homotrimeric properties of full-length CAPN3, it might be di fficult for termine whether three CAPN3 molecules can interact with one another PEF to preferentially form a heterotrimer with full-length CAPN3. These

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A B Fig. 1. BN-PAGE and density gradient analyses of SDS-PAGE BN-PAGE CAPN3. (A and B) Zero point five μg of lysates of COS7 cells COS7 sk muscle COS7 sk muscle transfected with empty vector (lane 1) or expression μ CSemp WT KI KO emp CS WT KI KO stomach vector for FLAG-CAPN3:C129S (lane 2), and 10 gof 12 3 45 12 3 45 6 skeletal muscle crude lysates of wild-type (lane 3), (kDa) Capn3 KI (lane 4), and Capn3 KO mice (lane 5) were subjected to SDS-PAGE (A) and BN-PAGE (B), both 175 – (kDa) followed by western blotting with anti CAPN3-IS2 antibody. Native CAPN3 and CAPN3:C129S, and 720 FLAG-CAPN3:C129S, are indicated by arrows. For 83 480 BN-PAGE analysis, detection of G-calpain, a ~160- 62 kDa gastrointestinal calpain heterodimer composed 242 of CAPN8 and CAPN9, from mouse stomach lysate 47 was performed as a positive control. G-calpain at the 146 position between the 146- and 242-kDa markers, detected using the anti-CAPN9 antibody, is indicated 32 66 by a closed arrowhead (lane 6). (C) Density gradient analysis of immunopurified FLAG-CAPN3:C129S complex. Upper, immunoblot of gradient fractions (10–50% OptiPrep, left to right) probed with anti- 2SI3NPAC-itna 2SI3NPAC-itna anti- FLAG to detect FLAG-CAPN3:C129S complex. Lower: CAPN9 the position of a 240-kDa marker, catalase, was vi- sualized by CBB-staining following SDS-PAGE. C 240 kDa FLAG-CAPN3:C129S complex (anti-CAPN3-IS2)

catalase (CBB stain) results also indicated the importance of additional region(s) for stable weight of 89 kDa as a monomer and 267 kDa as a trimer. These ob- homotrimer formation. servations are based on signals detected with the anti–CAPN3-IS2 an- tibody, which is more sensitive than the anti–CAPN3-NS antibody (Fig. 4B, lane 3 in both panels of BN-PAGE). From the samples sepa- 3.4. Autolyzed CAPN3 also forms a trimeric structure rated by BN-PAGE, signals corresponding to those detected with the anti–CAPN3-IS2 antibody were below the limit of detection of an- When CAPN3 is expressed in various expression systems including ti–CAPN3-NS antibody (Fig. 4B, lanes 1 and 2 in the right panel of BN- COS7 cells, it undergoes rapid autolysis in IS1 as a part of activation PAGE). However, we concluded that the signals detected using an- mechanism. Then, the resultant two autolytic fragments reconstitute a ti–CAPN3-IS2 were derived from the complex of the N- and C-terminal protease activity of CAPN3 by intermolecular complementation fragments, as the C-terminal fragment alone, which is identical to the (iMOC), a process unique to CAPN3 (Fig. 4A) [31]. Therefore, we in- 58 K deletion mutant, yielded a high molecular weight smear signal vestigated the quaternary structure of CAPN3 formed by iMOC upon (Fig. S1A, lane 6). Together, these results indicated that CAPN3 sustains activation. a trimeric structure under both pre- and post-autolysis conditions. When subjected to SDS-PAGE analysis, CAPN3 expressed in COS7 cells was barely detectable as autolytic fragments (Fig. 4B, lane 2 in the left panel of SDS-PAGE). In parallel, we analyzed a COS7 lysate ex- 4. Discussion pressing CAPN3 by BN-PAGE. Using anti–CAPN3-IS2 antibody, auto- lyzed CAPN3 was detected close to 242 kDa, a little lower than Previously, CAPN3 was described as being homodimeric, based on CAPN3:C129S (Fig. 4B, lanes 2 and 3 in the left panel of BN-PAGE). molecular modeling using the homodimeric 3D structure of the CAPN3 This is consistent with the idea that autolysis of CAPN3 clips out the PEF domain [32], and molecular weight estimation by gel filtration of majority of NS, as well as the IS1 region, resulting in a total molecular partially purified recombinant CAPN3:C129S [33]. In our study, how- weight of 258 kDa. These results suggested that CAPN3 forms a trimer ever, full-length CAPN3 expressed in cultured cells or extracted from after being translated, undergoes autolysis as a trimer, and remains as a mouse skeletal muscle lysates was detected as a homotrimer in several trimer after the autolysis. independent experiments. The discrepancy between our results and When the N-terminal and C-terminal fragments of CAPN3 are ex- those of previous studies implies the existence of different oligomeric pressed separately, it is also possible to reconstitute proteolytic activity states of CAPN3, which are sensitive to solution conditions. For ex- by iMOC (Fig. 4A) [31]. Hence, we investigated whether iMOC-medi- ample, p53, a tumor suppressor protein, whose oligomeric states are ated de novo CAPN3 reconstitution could be captured by BN-PAGE. As relevant to its regulation and activation, shifts from dimer to tetramer shown in Fig. 4B, when the N-terminal and C-terminal fragments (in upon stress signaling such as DNA damage in order to increase its DNA- which active site residues were mutated to avoid further autolysis) were binding activity [41]. Similarly, it is possible that CAPN3 converts its co-expressed in COS7 cells, a single signal was detected using an- oligomeric state between the homodimer and homotrimer depending ti–CAPN3-IS2 antibody that was intermediate in size between those of on cellular conditions. In line with this, oligomer formation and its CAPN3 and CAPN3:C129S (lanes 1–3 in the left panel of BN-PAGE). functional relevance (including functions independent of protease ac- This form of CAPN3, namely, inactive iMOC-CAPN3, lacks the IS1 re- tivity) have been described for some other proteases [44,45]. Further gion but retains NS, and is therefore expected to have a molecular investigation of the environmental factors related to such conversion

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A Sorcin CAPN3 GA (－) (＋) GA (－) (＋) 12 34 (kDa) (kDa) 190 315 135 250 100 175 75 140 58 95 46 72 32 52 25 22 43

anti-FLAG anti-FLAG

B FLAG-tagged MYC-tagged HA-tagged sample 1 CAPN3:C129S CAPN3:C129S empty vector 1st IP: anti-FLAG 2nd IP: anti-MYC sample 2 CAPN3:C129S empty vector CAPN3:C129S sample 3 CAPN3:C129S CAPN3:C129S CAPN3:C129S

input 2nd IP input 2nd IP input 2nd IP (kDa) 123123 123123 123 12 3 190 135 100 75 58

46

32

25

GALF-itna anti-MYC AH-itna

Fig. 2. CAPN3 forms a homotrimer. (A) FLAG-sorcin (lanes 1 and 2) or FLAG-CAPN3:C129S (lanes 3 and 4) was expressed in COS7 cells. Ten μg of the cell lysates incubated with (lanes 2 and 4) or without a final concentration of 0.05% GA (lanes 1 and 3) were analyzed by SDS-PAGE, followed by western blotting with anti-FLAG antibody. Crosslinked sorcin and CAPN3:C129S were indicated by closed arrowheads. (B) FLAG-, MYC-, and HA-CAPN3:C129S were co-expressed in COS7 cells in the indicated combinations. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates were subjected to a second immunoprecipitation with anti- MYC antibody. Anti-MYC antibody immunoprecipitates were analyzed by SDS-PAGE, followed by western blotting with anti-FLAG, anti-MYC, and anti-HA antibodies (2nd IP). Arrow indicates CAPN3:C129S fused with the epitope tags. could uncover novel aspects of CAPN3's function. that a trimeric form of CAPN3 is more efficient than a monomer or What is the significance of trimer formation? Upon trimerization, dimer form in performing proteolysis involved in a translocation. the number of active sites and protein-binding sites such as IS2 is tri- Two structural elements, the PEF and CBSW domains, are required pled, potentially increasing the efficacy of proteolytic activity or the for efficient trimer formation: simply removing the PEF domain causes strength of binding to muscle proteins. Skeletal muscles are the largest loss of oligomerization ability, and the CBSW domain is not sufficient as organs in the body, accounting for ~40% of body weight. In muscle an interface by itself, but indirectly allows trimerization through the cells, while CAPN3 resides in diverse cellular compartments, the ma- PEF domain. In our hands, the PEF domain demonstrated a strong jority (90–95%) of CAPN3 localizes to the myofibrils and the sarco- propensity to form dimers, which is consistent with the crystal structure plasmic reticulum (SR) [27,36]. In the myofibrils, CAPN3 is anchored [32], and had a trace avidity to full-length CAPN3. Because the latter to a giant myofibrillar protein, , via its N2A, M-line, and Z-line interaction was not detectable by BN-PAGE, stoichiometry of the regions. CAPN3 plays adaptive roles in titin-mediated mechanosensory components is unpredictable, and we assume that heterologous com- transduction such as muscle contraction or extension. In response to plex formation is a minor event. By contrast, the absence of the NS, IS1, muscle extension, CAPN3 rapidly alters its localization on titin and and IS2 does not affect trimer formation, consistent with previous re- accumulates at the N2A regions, which is facilitated by its proteolytic ports that these sequences are unstructured, have no homology to activity [23]. If CAPN3 forms a trimer, three catalytic sites are expected known structures, and could be allocated far from the interfaces per one functional unit of CAPN3. Therefore, it is tempting to speculate [46,47]. Upon activation, IS1 is released from each CAPN3 molecule

5 S. Hata, et al. BBA - Proteins and Proteomics 1868 (2020) 140411

A Fig. 3. The PEF domain is necessary but not sufficient for CAPN3 trimerization. (A) Schematic of the structures of full-length and de- letion mutants of human CAPN3 with N-terminal FLAG-tag used in this study. (B) FLAG-tagged full- C129S Full-length (aa. 1-821, 94kDa) FLAG- length (lanes 1, 2, 11, and 12) or the deletion mutants C129S of CAPN3 shown in (A) (lanes 3–10 and 13–20) were ΔEF5 (aa. 1-790, 90kDa) FLAG- C129S individually expressed in COS7 cells. Ten μg of cell ΔPEF (aa. 1-639, 74kDa) FLAG- C129S lysates were analyzed by SDS-PAGE and western ΔNS (aa. 49-821, 89kDa) FLAG- blotting using anti-FLAG antibody following treatment ΔNS-PC1 (aa. 235-821, 68kDa) FLAG- with (+, even lanes) or without (−, odd lanes) a final concentration of 0.05% GA. Closed and open arrow- 58K (aa. 323-821, 58kDa) FLAG- heads indicate the homotrimer and homodimer forms, Δ FLAG- NS-PC1/2 (aa. 429-821, 46kDa) respectively. (C) Full-length or deletion mutants of IS2+PEF (aa. 587-821, 27kDa) FLAG- CAPN3 were expressed in COS7 cells in combination PEF (aa. 641-821, 21kDa) FLAG- with MYC-CAPN3:C129S and HA- CAPN3:C129S. Cell lysates were subjected to immunoprecipitation with B anti-FLAG antibody, and immunoprecipitates were subsequently subjected to immunoprecipitation with anti-MYC antibody (2nd IP). The resultant im- Full-lengthΔEF5 ΔPEF ΔNS ΔNS-PC1 Full-length 58K ΔNS-PC1/2IS2-PEF PEF munoprecipitates were analyzed by SDS-PAGE and (GA) ー＋＋ ー ＋ー ＋ー ＋ー ＋ー ＋ー ＋ー ＋ー ＋ー western blotting with anti-FLAG, anti-MYC, and anti- fi 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 HA antibodies. Asterisks indicate non-speci c signals.

(kDa) (kDa) 250 250 150 150 100 100 75 75

50 50 37 37

25 25 20 20

anti-FLAG anti-FLAG C input 2nd IP

FLAG-tagged Full-lengthΔPEFΔNS ΔNS-PC158K ΔNS-PC1/2IS2-PEFPEF Full-lengthΔPEFΔNS ΔNS-PC158K ΔNS-PC1/2IS2-PEFPEF 12345678 910111213141516 (kDa) 190 135

* 100

75 FLAG-CAPN3:C129S or mutants 58 * 46

32 * 25

MYC-CAPN3:C129S

HA-CAPN3:C129S *

6 S. Hata, et al. BBA - Proteins and Proteomics 1868 (2020) 140411

Fig. 4. Trimer formation of iMOC-CAPN3. A (A) Schematic illustrations of activity regulation of NS IS IS CAPN3 through iMOC by its N-terminal (N-term) CAPN3 PC1 1 PC2 CBSW 2 PEF and C-terminal autolytic fragments (C-term). CAPN3 anti-CAPN3-NS anti-CAPN3-IS2 autolyzes in IS1, generating the N-terminal and C- autolysis in IS1 terminal fragments. The C-terminal fragment is identical to the 58 K deletion mutant. Both frag- ments are reconstituted by iMOC, leading to further NS IS PC1 PC2 CBSW 2 PEF autolysis. (B) The N- and C-terminal fragments were N-term (31kDa) C-term (= 58K deletion mutant, 58kDa) co-expressed in COS7 cells. Cell lysates were sub- iMOC jected to SDS-PAGE (left) and BN-PAGE (right), followed by western blotting with anti–CAPN3-IS2 and anti–CAPN3-NS antibodies (lane 1), and com- NS PC1 (31kDa) pared to those expressing FLAG-CAPN3 (lane 2) or FLAG-CAPN3:C129S (lane 3). Note that the N- IS (58kDa) PC2 CBSW 2 PEF terminal fragment used in this study harbored the C129S mutation and an N-terminal FLAG-tag. Open autolysis in the majority of NS iMOC-CAPN3 and closed arrowheads indicate the N-terminal and anti-CAPN3-NS C-terminal fragments, respectively. PC1 (28kDa) anti-CAPN3-IS2 IS PC2 CBSW 2 PEF (58kDa)

further autolysis

PC1 IS PEF CBSW 2 PC2

B SDS-PAGE BN-PAGE anti- anti- anti- anti- CAPN3-IS2 CAPN3-NS CAPN3-IS2 CAPN3-NS FLAG-CAPN3 - + - - - + - - + - FLAG-CAPN3:C129S --+ - --+ --+ N-term (31kDa) + -- + + -- + -- C-term (58kDa) + - - + + - - + - - (kDa) 1 32 11(kDa) 23 123 190 1048 135 100 720 75 480 58 242 46 146 32 66

followed by protease domain reconstitution by iMOC, while the C- Declaration of competing interest terminal CBSW and PEF domains anchor three molecules in the trimeric state. Further studies are required to fully elucidate the intermolecular The authors declare that they have no conflicts of interest related to interactions for trimer formation. the contents of this article. In conclusion, using a combination of biochemical and biophysical approaches, we obtained evidence that CAPN3 forms a homotrimer. Acknowledgments This study is the first to describe trimer formation by a calpain isoform as well as PEF family proteins. Also, the present results will contribute This paper is respectfully dedicated to our mentor, Dr. Hiroyuki to understanding the pathogenesis of LGMD2A in terms of how CAPN3 Sorimachi, who passed away on January 2018. We thank all of the is inactivated by its pathological mutations. laboratory members for valuable discussions. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbapap.2020.140411. Author contributions

S.H. designed, performed, and analyzed the experiments, and wrote the paper. N.D. provided technical assistance and contributed to the

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