Cloning of a muscle-specific calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting

A cDNA (1977 bp) encoding a crustacean calpain (Ha-CalpM; GenBank accession no. AY124009) was isolated from a lobster fast muscle cDNA library. The open reading frame specified a 575-amino acid (aa) polypeptide with an estimated mass of 66.3 kDa. Ha-CalpM shared high identity with other calpains in the cysteine proteinase domain (domain II; aa 111-396) and domain III (aa 397-575), but most of the N-terminal domain (domain I; aa 1-110) was highly divergent. Domain II contained the cysteine, histidine and asparagine triad essential for catalysis, as well as two conserved aspartate residues that bind Ca(2+). In domain III an acidic loop in the C2-like region, which mediates Ca(2+)-dependent phospholipid binding, had an expanded stretch of 17 aspartate residues. Ha-CalpM was classified as a non-EF-hand calpain, as it lacked domain IV, a calmodulin-like region containing five EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly expressed in skeletal muscles, but at much lower levels in heart, digestive gland, intestine, integument, gill, nerve cord/thoracic ganglion and antennal gland. An antibody raised against a unique N-terminal sequence recognized a 62 kDa isoform in cutter claw and crusher claw closer muscles and a 68 kDa isoform in deep abdominal muscle. Ha-CalpM was distributed throughout the cytoplasm, as well as in some nuclei, of muscle fibers. Purification of Ha-CalpM showed that the 62 kDa and 68 kDa isoforms co-eluted from gel filtration and ion exchange columns at positions consistent with those of previously described Ca(2+)-dependent proteinase III (CDP III; 59 kDa). Ha-CalpM mRNA and protein did not change during the moulting cycle. The muscle-specific expression of Ha-CalpM and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins suggest that it is involved in restructuring and/or maintaining contractile structures in crustacean skeletal muscle.