Three calpains and ecdysone receptor in the land crab Gecarcinus lateralis: sequences, expression and effects of elevated ecdysteroid induced by eyestalk ablation

Abstract

SUMMARY Crustacean muscle has four calpain-like proteinase activities (CDP I, IIa,IIb and III) that are involved in molt-induced claw muscle atrophy, as they degrade myofibrillar proteins in vitro and in situ. Using PCR cloning techniques, three full-length calpain cDNAs (Gl-CalpB, Gl-CalpM and Gl-CalpT) were isolated from limb regenerates of the tropical land crab Gecarcinus lateralis. All three had highly conserved catalytic (dII)and C2-like (dIII) domains. Gl-CalpB was classified as a typical,or EF-hand, calpain, as the deduced amino acid sequence had a calmodulin-like domain IV in the C-terminus and was most similar to Drosophilacalpains A and B. Based on its estimated mass (∼88.9 kDa) and cross-immunoreactivity with a polyclonal antibody raised against Dm-CalpA,Gl-CalpB may encode CDP IIb, which is a homodimer of a 95-kDa subunit. It was expressed in all tissues examined, including skeletal muscle, heart,integument, gill, digestive gland, hindgut, nerve ganglia, gonads and Y-organ(molting gland). Both Gl-CalpM and Gl-CalpT were classified as atypical, or non-EF-hand, calpains, as they lacked a domain IV sequence. Gl-CalpM was a homolog of Ha-CalpM from lobster, based on similarities in deduced amino acid sequence, estimated mass (∼65.2 kDa) and structural organization (both were truncated at the C-terminal end of dIII). It was expressed at varying levels in most tissues, except Y-organ. Gl-CalpT (∼74.6 kDa) was similar to TRA-3 in the nematode Caenorhabditis elegans; domain IV was replaced by a unique `T domain' sequence. It was expressed in most tissues,except eyestalk ganglia and Y-organ. The effects of elevated ecdysteroid,induced by eyestalk ablation, on calpain and ecdysone receptor (Gl-EcR) mRNA levels in skeletal muscles were quantified by real-time PCR. At 1 day after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels increased 15- and 19.3-fold, respectively, in claw muscle but not in thoracic muscle. At 3 days after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels in claw muscle had decreased to 2.8-fold and 4.3-fold higher than those in intact controls,respectively, suggesting a feedback inhibition by ecdysteroid. There was no significant effect of eyestalk ablation on Gl-CalpB and Gl-CalpM mRNA levels. Gl-CalpT and Gl-EcR mRNA levels were significantly correlated in both claw and thoracic muscles from intact and eyestalk-ablated animals. The data suggest that Gl-CalpT is involved in initiation of claw muscle atrophy by ecdysteroids. Premolt reduction in claw muscle mass and concomitant remodeling of the sarcomere probably result from post-transcriptional regulation of calpains.