Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

Figure 1. Abnormal NH2-terminal processing of the LGMDR1 mutants of CAPN3 within the PEF domain.A, schematic illustration of CAPN3 and its LGMDR1 mutants within the PEF domain. Stars indicate the cleavage sites recognized by the anti-AIS1 antibody. The bar indicates the antigen region (488–666 amino acids encoded by BC146672) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 28476-1-AP). . C129, H334, and N358 are catalytic centers. Arrows indicate autolytic cleavage sites. B, Western blotting of the cell lysates transfected with WT, protease-inactive form (C129S), and LGMDR1 mutants of EGFP-CAPN3 with anti-CAPN3 (28476-1-AP, upper panel) and anti-AIS1 (lower panel) antibodies. The cell lysates of CAPN3:CS lack the two AIS1-recognizing fragments. C, band intensity ratio of “fragment b” to “fragment a” in the LGMDR1 mutants within PEF domains. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolysis within IS1; NS, N-terminal sequence; IS, internal sequence; PC, protease core; CBSW, calpain-type β-sandwich; PEF, penta-EF motif; LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain.

Figure 2. Oligomerization of LGMDR1 mutants of CAPN3 within the PEF domain.A, Western blotting of LGMDR1 mutant forms of EGFP-CAPN3 with (right panel) or without (left panel) cross-linking by 0.1% glutaraldehyde. B, ratio of the band intensity of oligomer (>250 kDa) to monomer (∼130 kDa) forms of the LGMDR1 mutants within the PEF domain. ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. C, 3D structural model of the CAPN3 dimer and position of the I807 amino acid residue (red) within the EF5 loop (Protein Data Bank ID: 4OKH) (13). D, oligomerization of CAPN3:CS and CAPN3:CS:I807T expressed in HEK293T cells. E, relative ratio of oligomer to monomer. ∗p = 0.0121, Mann–Whitney U test. F, Western blotting of the cell lysates expressing WT and the I807T mutant form of EGFP-CAPN3 with anti-CAPN3 (upper panel) and anti-AIS1 (lower panel) antibodies. G, band intensity ratio of “fragment b” to “fragment a” of EGFP-CAPN3:I807T. ∗∗∗p = 0.00084, Mann–Whitney U test. H, scatter plot of the oligomer/monomer ratio against the NH2-terminal processing ratio (b/a) of various LGMDR1 mutants within the PEF domain. Mutants in red lack “fragment b” when expressed in HEK293T cells.…

Figure 3. Analysis of CAPN3 binding to the two titin regions in cells with heterologous overexpression.A, schematic illustration of the mCherry-fusion proteins with the two regions of human titin (NCBI reference sequence: NP_596869.4). The N2A region corresponds to the mouse mCN48 fragment identified as a p94-binding fragment via yeast two-hybrid screening in our previous study (25). B, EGFP-CAPN3:CS is coimmunoprecipitated with mCherry-titin CT more efficiently than with mCherry-titin I80-PEVK in HEK293T cells. Because of their weak interaction, the cells were treated with a dithiobis(succinimidyl propionate) cross-linker before cell lysis and immunoprecipitation. C, band intensity ratio of the coimmunoprecipitated CAPN3 to the CAPN3 in the lysate. ∗p = 0.0211, Mann–Whitney U test. D, immunostaining of EGFP-CAPN3:CS (green) coexpressed with mCherry fusion proteins of the two titin regions (magenta) in HeLa cells. Arrows (upper panel) and arrowheads (bottom panel) show filopodia with no GFP staining. The scale bar represents 20 μm. E, translocation of EGFP-CAPN3:CS to the membrane fractions by mCherry-titin CT-kRas, but not mCherry-titin I80-PEVK-kRas, in HeLa cells. HeLa cells were…

Figure 4. Localization of LGMDR1 mutants of EGFP-CAPN3 and mCherry-titin CT in HeLa cells.A, immunostaining of EGFP-CAPN3:CS (green) and its LGMDR1 mutants coexpressed with mCherry-titin CT (magenta) in HeLa cells. The scale bar represents 10 μm. B, quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT. Pearson correlation coefficient was calculated using Coloc2 software. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; CT, COOH terminal.

Figure 5. Localization of EGFP-CAPN3 in TA muscles of Capn3−/− mice following AAV-mediated expression.A, immunohistochemistry of AAV-infected EGFP, EGFP-CAPN3, EGFP-CAPN3:CS, EGFP-CAPN3:CS:DIS2 (GFP, green), and actinin (magenta) in Capn3−/− mice (left panels). Right panels show intensity plots of EGFP and actinin immunoreactivity along the line in each merged image. The scale bar represents 10 μm. B, immunohistochemistry of endogenous CAPN3 and actinin in the gastrocnemius muscle of 12-week-old Capn3+/+ and Capn3−/− mice. Tissues were stained with anti-CAPN3 (green, Proteintech 28476-1-AP) and anti-actinin (magenta) antibodies (left panels). Right panels indicate the line plot of CAPN3 and actinin immunosignals within the merged images. The scale bar represents 10 μm. CAPN, calpain; EGFP, enhanced green fluorescent protein; AAV, adeno-associated virus; CT, COOH terminal; TA, tibialis anterior.

Figure 6. Subcellular localization of LGMDR1 mutants of EGFP-CAPN3:CS in Capn3−/− mice TA following AAV-mediated expression.A–C, immunohistochemistry of the AAV-infected LGMDR1 mutant form of EGFP-CAPN3:CS (anti-GFP antibody, green) and anti-actinin (magenta) antibody (left panels) in Capn3−/− mice. Right panels show the intensity plot of EGFP and actinin immunoreactivity along the line in each merged image. Three patterns of CAPN3 immunosignals were observed in the LGMDR1 mutants within the PEF domain: fine M-band signals (A), broadened M-band signals (B), and signals around the N2A region of sarcomeres (C). The scale bar represents 10 μm. LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; AAV, adeno-associated virus; TA, tibialis anterior; PEF, penta-EF hand.

Figure 7. Partial rescues of abnormal intracellular localization of EGFP-CAPN3:CS:A702V but not of EGFP-CAPN3:CS:D705H in Capn3+/+ mice.A, immunostaining of actinin (magenta) and EGFP-CAPN3:CS:A702V and EGFP-CAPN3:CS:D705H (green) in the TA muscles of Capn3+/+ mice. Right panel shows the line plot of the merged image. The scale bar represents 10 μm. B, coexpression of CAPN3:CS ameliorated the localization of EGFP-CAPN3:CS:A702V but not EGFP-CAPN3:CS:D705H at the mCherry-titin CT (magenta) enriched plasma membrane in HeLa cells. Right panels show the line plot of mCherry-titin CT (magenta) and EGFP-CAPN3:CS, CAPN3:CS:A702V, and CAPN3:CS:D705H (green) within the left merged images. C, quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT upon coexpression of CAPN3 in HeLa cells. Pearson correlation coefficient was calculated using Coloc2 software. ∗∗∗p = 0.0019, one-way ANOVA with Bonferroni multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CT, COOH terminal; CAPN, calpain; EGFP, enhanced green fluorescent protein; TA, tibialis anterior.