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.Study of calpain 3 received an important impetus after the demonstration of its involvement in limb girdle muscular dystrophy type 2A (Mendelian Inheritance in Man [MIM] 253600) (24). This neuromuscular disorder is characterized mainly by symmetrical atrophy and weakness of proximal limb muscles, by elevated creatine kinase in serum, and by a dystrophic pattern in muscle biopsies (4). Calpains are members of a family of intracellular nonlysosomal cysteine proteases (for reviews, see references 35 and 36). They are comprised of three ubiquitous calpains (, m, and /m); a skeletal musclespecific calpain (calpain 3, CAPN3, nCL-1, or p94 [31]), a variant of which is also expressed in a lens-specific manner (17, 18); a digestive tract-specific calpain ); and the stomach-specific calpains (nCL-2 and nCL-2Ј [33]).The human calpain 3 gene was reported to consist of 24 exons spanning approximately 45 kb (24). It encodes a 3.5-kb mRNA expressed predominantly in skeletal muscle tissues. The 821-amino-acid-long calpain 3 protein can be subdivided, like the other calpains, into four domains that include a proteolytic (domain II) and a calcium binding (domain IV) domain (26, 31). In addition, three short calpain 3-specific sequences (NS, IS1, and IS2 [36]) are present. These are located, respectively, at the N terminus, in the protease domain, and between domains III and IV. IS2 includes a titin (connectin) binding site (11, 34) as well as a putative nuclear localization signal (31). Calpain 3 differs from the ubiquitous calpains by its rapid autolysis, at least when it is expre...

Stable expression of calpain 3 from a muscle transgene in vivo : Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

“…The ex 6Ϫ Tg showed no activity above non-Tg control muscle, even though expression levels of this transgene were approximately 60-fold higher than endogenous C3. Because it has been demonstrated in vitro that ex 6Ϫ constructs retain some proteolytic activity, it cannot be concluded that all C3 activity is abolished when this transgene is overexpressed (5). However, these data suggest that extracts from ex 6Ϫ Tg may have reduced C3 proteolytic activity compared with non-Tg and show that extracts from mice ex- pressing full-length C3 and ex 15Ϫ Tg produce higher C3 activity than extracts from non-Tg mice.…”

“…In addition, overexpression of calpastatin, the calpainspecific inhibitor (9,10), also blocks muscle cell differentiation in vitro. Furthermore, alternatively spliced C3 isoforms lacking portions of either IS1and͞or IS2 have been observed in developing human and mouse skeletal muscle by using reverse transcription-PCR (5). After birth, expression levels of the deletion isoforms decline and the full-length, mature isoform predominates.…”

“…C3 is an extremely unstable protein that has not been successfully purified because of its ability to autolyze rapidly (4). Expression of the protein from constructs in vitro has also been extremely inefficient, unless the active site is mutated or truncations of certain exons (ex) are performed (3,5). In addition, purification in a baculovirus system has only succeeded in producing a truncated C3 (6).…”

Myogenic Stage, Sarcomere Length, and Protease Activity Modulate Localization of Muscle-specific Calpain

“…In transgenic mice overexpression of p94⌬ex6 or p94⌬ex15, but not full-length p94WT, blocked/delayed maturation of myofibers (34). These variants, which were shown to be less active than p94 especially with regard to autolytic activity (29), could act detrimentally at various developmental stages. Thus, these previous data and our data are in support that both the transient expression and the disappearance of p94 isoforms at the early stages of myogenesis are important for myotube maturation, probably because these isoforms respond differently to the cellular mechanisms regulating p94.…”

“…Because previous studies have revealed that mouse embryonic muscles express p94 variants without exons 6, 15, and/or 16 (29), the relative abundance of transcripts resulting from those exon-skipping events was examined (Fig. 1A).…”