Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events

Abstract

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

FIG. 1

Electrophoresis of variant PCR products from the systematic screening of myoblast and myotube RNAs. Amplifications were performed with p94sys3 primers flanking the IS1 region (A) and with p94sys5 (B) and p94sys6 (C) primers flanking the IS2 region. MB, myoblast; MT, myotube. (A) The 396-bp band corresponds to the splicing out of exon 6. (B) The 601-, 505-, and 487-bp bands correspond, respectively, to the skipping of exon 15 and exon 16 and the splicing out of both exons 15 and 16. (C) The 603- and 774-bp bands correspond, respectively, to the retention of the int137 and int308 sequences.

FIG. 2

Alternative splicing events of mouse calpain 3 mRNA. (A) Schematic diagrams of the calpain 3 protein with its four domains and its three specific sequences, NS, IS1, and IS2, and the exon structure of the mRNA. Positions of the primers used for long-range PCR are indicated. (B) Sites of alternative splicing with deletion of (i) exon 6, (ii) exon 15, (iii) exon 16, and (iv) exons 15 and 16. Arrows denote the positions of primers used in PCR for identifying alternative splicing events. (C) Variant forms of mouse calpain 3 mRNA produced by the association of alternative splicing of the three exons, 6, 15, and 16 (gaps in black lines), and retention of the sequences int137 and int308 (short and somewhat longer gray lines, respectively). (D) Retention of the sequences int137 and int308 in intron 16. AS and DS are 3′ acceptor and 5′ donor sites, respectively. Putative splice sites are in italics. The scores of comparison between consensus splice sites and normal or putative alternative splice sites are indicated.

FIG. 3

Visualization of calpain 3 isoforms. Electrophoresis of RT-PCR products during mouse embryonic development from days 11.5 to 18.5 (A) and in different tissues (embryonic lens and lens, brain, heart, and smooth and skeletal muscle tissues from 9- to 11-week-old mice) (B). E, embryonic day. (a) PCR products obtained with p94sys3 primers. The 540- and 396-bp bands correspond, respectively, to the entire segment with and without exon 6. (b) PCR products obtained with primers p94sys5.a and p94sys5b.m. The 619-bp band is from the unspliced mRNA. The 601-, 505-, and 487-bp bands correspond, respectively, to the splicings of exon 15 and exon 16 and the association of splicings of exons 15 and 16. The embryonic lens profile was obtained with a Southern blot probed with the peroxidase-labeled primer sIS2 (5′-GGAAGCTGAAAATACAATCTCTG-3′, positions 1746 to 1768) (the figure is a photographic negative). (c) Amplification of a TFIID sequence.

FIG. 4

Measure of calpain 3 mRNA level by quantitative RT-PCR. Calpain 3 transcription was monitored in six different tissues by a quantitative PCR method with TaqMan probes. The level of TFIID mRNA was used to normalize the results across different tissues. An arbitrary value of 1 was assigned to the skeletal muscle value, and the values measured in the other tissues are expressed as ratios to the skeletal muscle content, these ratios being drawn in a logarithmic scale.

FIG. 5

In situ hybridization on an embryonic lens region. Shown are transverse sections of NMRI mice embryos at day 12.5 (A to C) and sagittal sections at day 17.5 (D to F). In the phase-contrast images (A and D), the bars represent 600 mm. The other panels show results of dark-field in situ hybridization with a probe specific for the exon 1′ sequence (var5pAS) (B and E), a sense probe (var5p.S) (F), and a titin-specific probe (C) (6). EOM, extrinsic ocular muscle; FM, facial muscle; L, lens, NL, neural layer of retina; O, orbital plate of frontal bone; PNC, primitive nasal cavity; PV, primitive vitreous humor. Note that pigments of the neural layer of the retina and primitive ossification within the medial part of the roof of the orbit are due to nonspecific labeling.

FIG. 6

Investigation of the autolytic and fodrinolytic capacities and titin binding abilities of the different isoforms. (A) The isoforms investigated for biochemical characteristics are drawn under the diagram of the calpain 3 protein (Lp82 corresponds to the ex1′⁺,1⁻6⁻15⁻16⁻ isoform). Their names and molecular masses are given at the left of the line. The position of the C129S mutation, which inactivates the catalytic site, is indicated. Western blot analyses were performed on lysates of transfected COS-7 cells to visualize proteolytic calpain 3 fragments with an antibody against the IS2-specific region of p94 (B) and to assess fodrinolysis with an antibody specific to the 150-kDa α-fodrin fragment (C). Open arrowheads indicate the full-length products, and filled arrowheads indicate proteolyzed fragments. The titin binding capacity of each isoform was monitored in a yeast two-hybrid system by measuring β-galactosidase activity with CPRG as the substrate (1 U of β-galactosidase is defined as the amount which hydrolyzes 1 mmol of CPRG in 1 min). (D and E) Histograms of β-galactosidase expression for each isoform following cotransfection into S. cerevisiae cells of a calpain 3 isoform and pCNT-N2 (D) and pCNT-52 (E) titin peptide-encoding clones. Bars indicate the mean β-galactosidase activities from three independent experiments; ranges are indicated by the vertical lines. (Lp82 corresponds to the ex1′⁺,1⁻6⁻15⁻16⁻ isoform). The first column corresponds to transfection with the vector pAS2C-1 alone.