Yu, X.L.*; Mykles, D.L.: Cloning and Characterization of Skeletal Muscle Specific Calpain from Lobster (Homarus americanus)
Crustacean muscles contain four calcium-dependent cysteine proteinases (CDPs or calpains) that differ in mass and subunit composition. These enzymes (CDP I, IIa, IIb, and III) are involved in the degradation of myofibrillar proteins during a programmed atrophy associated with molting. We have begun cloning these genes to analyze their expression in various tissues during the intermolt cycle. Highly degenerate universal calpain primers were designed based on the homologous amino acid sequences from multiple alignments of calpains from diverse species. Using nested PCR and inverse PCR, a full-length (1978 bp) lobster calpain gene (Ha-CalpB) was isolated and sequenced from a deep abdominal muscle cDNA library. The deduced amino acid sequence of the polypeptide (about 55 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 about 230 bp; domain II consists of a highly-conserved cysteine protease sequence; and domain III contains a unique stretch of 17 aspartates and a highly-conserved Ca2+ -binding region. Ha-CalpB, like Dm-CalpB, lacks a calmodulin-like sequence (Domain IV) found in the ubiquitous m-calpain and �-calpain in mammals. Northern blot and RT-PCR revealed that Ha-CalpB is highly expressed in skeletal muscle, with little or no expression in other tissues. A polyclonal antibody raised against a unique amino acid sequence (residues 53-80) in Domain I is being used to identify which of the four lobster calpains is encoded by Ha-CalpB. CDP IIa (native Mr = 125 kDa; subunit Mr = 60 kDa) and/or CDP III (native Mr = 59 kDa; subunit composition unknown) are potential candidates. Supported by NSF (IBN-9904528).
