YU, Xiaoli; MYKLES, Donald: ISOLATION AND CHARACTERIZATION OF A SKELETAL MUSCLE SPECIFIC CALPAIN FROM LOBSTER (Homarus americanus)

Crustacean muscles contain four calcium-dependent cysteine proteinases (CDPs or calpains) which are involved in the degradation of myofibrillar proteins during a programmed atrophy associated with molting. Using nested PCR and inverse PCR, a full-length (1978 bp) lobster calpain gene (Ha-CalpM) was isolated and sequenced from a deep abdominal muscle cDNA library. The deduced amino acid sequence of the polypeptide (575 aa, 66.3 kDa) has high sequence identity to other calpains, particularly the Drosophila Calpain B (Dm-CalpB) gene. Domain I has a unique N-terminal sequence of 110 aa; domain II consists of a highly-conserved cysteine protease sequence with the conserved catalytic triad-Cys 141, His 299 and Asn 329; and domain III contains an extremely acidic region (an expanded stretch of 17 aspartates) which may bind Ca²⁺ and function as an “electrostatic switch” involved in Ca²⁺– dependent activation. Ha-CalpM, like Dm-CalpA’ lacks a calmodulin-like domain (Domain IV) found in the ubiquitous m-calpain and �-calpain in mammals. Gene expression analysis revealed that Ha-CalpM is highly expressed in skeletal muscle, with little or no expression in other tissues. Real-time PCR showed that it is up regulated during the premolt stage in claw muscles undergoing atrophy. A polyclonal antibody raised against a unique amino acid sequence (residues #53-80) in Domain I was used to determine the location and relative amounts of Ha-CalpM protein expressed in lobster tissues. Immunocytochmistry indicated that Ha-CalpM is localized in both the cytoplasm and nuclei of muscle fibers. Supported by NSF (IBN-9904528).