Disease relevance of CAPN3

• Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy [1].
• Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3 [1].
• The Ttn(mdm) mutant mouse may serve as a model for human tibial muscular dystrophy, which maps to the TTN locus at 2q31 and shows a secondary reduction of CAPN3 similar to that observed in mdm skeletal muscle [2].
• However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells [3].
• By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression [4].

High impact information on CAPN3

• The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3 [5].
• Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A [6].
• The autosomal recessive forms (LGMD2) constitute a genetically heterogeneous group with LGMD2A mapping to chromosome 15q15.1-q21 [6].
• Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles [7].
• Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients [7].

Chemical compound and disease context of CAPN3

• We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a recombinant protein in the absence of Ca(2+) and undergoes autolysis in its presence [8].
• The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A [9].
• In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment [10].

Biological context of CAPN3

• Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction [1].
• Correlations between the nature and site of a particular mutation and its corresponding phenotype, however, can only be established from homozygous mutations, which are particularly rare in the alternatively spliced NS, IS1 and IS2 regions of CAPN3 [11].
• The 550delA mutation was present on 76% of CAPN3 chromosomes that led us to screen general population for this mutation; 532 random blood samples from three different regions were analyzed using allele-specific PCR [12].
• While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein [4].
• To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated [13].

Anatomical context of CAPN3

• Calpain3 (CAPN3) RNA was found in only skeletal muscles during the fetal period [14].
• These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling [13].
• In myofibrils, p94 specifically binds to connectin/titin, and the activity of p94 is probably suppressed by this binding [15].
• In contrast to the ubiquitous expression of mu- and m-types, Northern blot analysis revealed that the mRNA for p94 exists only in skeletal muscle with none detected in other tissues including heart muscle and smooth muscles such as intestine [16].
• 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 [17].

Associations of CAPN3 with chemical compounds

• An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography [3].
• p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains [18].
• Muscle-specific calpain, p94, interacts with the extreme C-terminal region of connectin, a unique region flanked by two immunoglobulin C2 motifs [19].
• Patients presented with a triad that appears to be indicative of CAPN3 mutations: (1) EM in the first decade, (2) elevated serum creatine phosphokinase levels (isolated or with little corresponding weakness), and (3) inconstant peripheral hypereosinophilia [20].
• RESULTS: In human subjects, calpain 3 gene expression was negatively correlated with total (P = 0.022) and central abdominal fat mass (P = 0.034), and with blood glucose concentration in non-obese subjects (P = 0.017) [21].

Physical interactions of CAPN3

• Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei [10].

Regulatory relationships of CAPN3

• For example, deficiency of dysferlin disrupts sarcolemmal membrane repair, whilst loss of calpain-3 may exert its pathological influence either by perturbation of the IkappaBalpha/NF-kappaB pathway, or through calpain-dependent cytoskeletal remodelling [22].

Other interactions of CAPN3

• Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family [3].
• Seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: from LGMD2A to LGMD2G [23].
• We have developed fluorescent genetic markers bracketing six of these loci (LGMD2A-LGMD2F) [24].
• The loss of function of a calpain species eventually leads to the activation of proteases including other calpain species responsible for muscle degradation. p94 does not form a complex with the small subunit of calpain (30K), but exists as a homodimer [25].
• Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD) [26].

Analytical, diagnostic and therapeutic context of CAPN3

• To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated [1].
• Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems [3].
• MEASUREMENTS: Expression of CAPN3 in skeletal muscle was measured using Taqman fluorogenic PCR [21].
• These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody [3].
• CONCLUSIONS: A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken [27].

References