Yasuko Ono

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.

Possible Regulation of the Conventional Calpain System by Skeletal Muscle-specific Calpain, p94/Calpain 3

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a “modulator protease” in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94:exons 6–15–16– (p94Δ), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca2+-independent autolytic activity, p94Δ was stably expressed in COS and insect cells. p94Δ showed Ca2+-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94Δ and is a substrate for p94Δ, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94Δ, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.

Skeletal Muscle-Specific Calpain, p94, and Connectin/Titin: Their Physiological Functions and Relationship to Limb-Girdle Muscular Dystrophy Type 2A

The skeletal muscle-specific calpain homologue, p94 (also called calpain 3), is essential for normal muscle function. A mutation of the p94 gene causes limb-girdle muscular dystrophy type 2A (LGMD2A), which is one type of autosomal recessive inherited disease characterized by progressive muscular degeneration. In myofibrils, p94 specifically binds to connectin/titin, and the activity of p94 is probably suppressed by this binding. Thus, we postulate that a signal transduction pathway exists, involving p94 and connectin/titin to modulate functions of skeletal muscle, and LGMD2A occurs when this signalling pathway is not properly regulated by p94. LGMD2A mutants of p94 also reveal significant information on the factors that relate structure to function in this molecule.

Newly identified exons encoding novel variants of p94/calpain 3 are expressed ubiquitously and overlap the α-glucosidase C gene

There are two classes of an intracellular ‘modulator protease’, calpain: ubiquitous and tissue‐specific. p94/calpain 3 is an example of the latter, predominantly expressed in muscle. A defect in the p94 gene causes muscular dystrophy. Here we report that human and mouse p94 genes have a possible novel alternative promoter expressing p94 variants in all tissues examined including human lens epithelial cells. The possible promoter region and the following novel exons overlap the 3′ region of the neutral α‐glucosidase C gene. Unlike p94, the novel p94 variants expressed in COS7 cells do not undergo rapid autolysis, suggesting basic functions different from p94.

New Aspect of the Research on Limb-Girdle Muscular Dystrophy 2A: A Molecular Biologic and Biochemical Approach to Pathology

p94, a muscle-specific member of the calpain family, also called calpain3 (CAPN3), has been identified as the gene product responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). To elucidate the molecular mechanism of LGMD2A, the effects of missense point mutations found in LGMD2A on the unique properties of p94 were studied. All of the mutants examined to date lose their proteolytic activity against fodrin, a cytoskeletal protein, strongly suggesting that of the specific properties of p94, the loss of protease activity is the prime cause of LGMD2A. Studies of LGMD2A and p94 suggest a novel molecular mechanism for muscular dystrophy, showing that a combined pathologic and biochemical approach is effective.

Calcium and Muscle Disease: Pathophysiology of Calpains and Limb-Girdle Muscular Dystrophy Type 2A (LGMD2A)

Precise regulation of numerous cellular events is essential for cell function and survival. Proteolysis in the cytosol is one of the important systems that modulate cellular functions internally. In muscle tissues, the involvement of several classes of proteases in various cellular events has been studied extensively, including their roles in myofibrillar protein turnover, myoblast fusion, cell cycle progression, and so on. Calpain, an intracellular Ca2+-dependent cysteine protease, is considered one of the most significant proteases in muscle tissues in both normal and pathological conditions. Muscular dystrophy is a group of neuromuscular disorders that present with necrosis and irregularly sized muscle fibers. Excessive degradation of myofibrillar proteins is responsible for this pathological change. Since degenerative muscle is often accompanied by an elevated cytosolic Ca2+ concentration, it is almost certain that proteolysis by calpain initiates the fiber necrosis. Therefore, the study of calpain in muscular dystrophy has aimed principally at developing means of suppressing calpain activity. Recent studies have revealed another aspect of the relationship between calpain and muscular dystrophy. A mutation in p94, also called calpain 3, is responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). This provides definitive evidence that the calpain system is vital for normal muscle function. Moreover, it suggests for the first time that the activity of one calpain species might oppose the effect of another. In this chapter, we briefly review the molecular structure of calpain and focus on the uncertain, but likely, pathophysiological roles of calpain in muscle diseases based on current knowledge.

Corrigendum to: Newly identified exons encoding novel variants of p94/calpain 3 are expressed ubiquitously and overlap the α‐glucosidase C gene (FEBS 27898)

aLaboratory of Biological Function, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan bCREST, JST, Kawaguchi 332-0012, Japan cSchool of Pharmaceutical Sciences, Kinki University, Higashi-Osaka 577-8502, Japan dNew Frontiers Research Laboratories, Toray Industries Inc., Kamakura 248-8555, Japan

Calpains: structure and function of the calpain super family

Calpain (EC 3.4.22.17) is a typical cytosolic cysteine protease whose activity is regulated by calcium, an important second messenger of extra-cellular stimuli [1–4]. Since calpain is distributed ubiquitously among cells and tissues of most higher organisms, it has been predicted to play an essential role in cellular functions. One reason that calpain attracts such interest is its way of hydrolyzing substrates; i.e. calpain may modulate, by limited proteolysis, the function of substrate proteins. Although, the physiological function of calpain as a biomodulator remains to be clarified, recent technological developments enable a variety of new approaches. These include finding tissue-specific calpain species, calpain-homologues in lower organisms, and the introduction of new methodology. In this chapter, the state of the art of calpain is summarized.