Three calpains and ecdysone receptor in the land crab Gecarcinus lateralis: sequences, expression and effects of elevated ecdysteroid induced by eyestalk ablation.
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 Drosophila calpains A and B. Based on its estimated mass (approximately 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 (approximately 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 (approximately 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.
Kim
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HW
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
Chang
E S
ES
Mykles
D L
DL
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics* / Q000378:metabolism; D002154:Calpain / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D026461:Ecdysteroids / Q000378:metabolism*; D040461:Gene Components; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology; D011647:Puerto Rico; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D026461:Ecdysteroids; D011987:Receptors, Steroid; C055469:ecdysone receptor; D002154:Calpain