Characterization of genes involved in molting and limb regeneration in land crab, Gecarcinus lateralis

Abstract

The claw muscles of decapod crustaceans undergo a programmed atrophy to facilitate withdrawal of the claws at the time of molting. The muscle has four calpain-like proteinase activities (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins. Three calpain cDNAs were isolated (Gl-CalpM, -B, and -T) using nested polymerase chain reaction (PCR) and 3' and 5' rapid amplication of cDNA ends (RACE) PCR. Gl-CalpM is a homolog of lobster calpain M (Ha-CalpM), as determined by deduced amino acid sequence and estimated mass (~6 8 kDa). It is expressed at high levels in skeletal muscle and ovary. Gl-CalpB contains all four domains (I, II, II, and IV) of typical calpains and has highest homology to Drosophila calpains A and B (Dm-CalpA and B). Based on its estimate mass (~89 kDa) and cross immunoreactivity with a polyclonal antibody raised against Dm-CalpA, the Gl-CalpB appeared to encode CDP IIb, which is a homodimer of a 95-kDa subunit. Gl-CalpT is a homolog of nematode TRA-3 and human calpain 5, which contain a unique "T" domain in place of the N-terminal EF-hand domain IV of typical calpains. Both Gl-CalpB and Gl-CalpT were expressed in a wide variety of tissues at varying levels. Since muscle atrophy coincides with increased ecdysteroid concentrations in the hemolymph, the molting hormone, 20-hydroxyecdysone (20E), may be involved, either directly or indirectly, in activating calpain-mediated myofibrillar protein degradation. Using nested PCR and RACE, a partial sequence of the ecdysone receptor (Gl-EcR), a full-length sequence of retinoid receptor (Gl-RXR), and a full-length sequence of an ecdysone early-response gene (G1-E75) were isolated. Gl-EcR was expressed as a single isoform, while Gl-RXR was expressed as 7 isoforms generated by alternative splicing. As the functional 20E receptor is a heterodimer of EcR and RXR, the different Gl-RXR isoforms may confer different functional properties for tissue responses to the same level of hormone. Gl-E75 has the highest sequence identity to shrimp E75 (Me-E75), although the F domain in Gl-E75 was more similar to that of Drosophila E75 (Dm-E75). Quantitative real-time PCR showed that expression of EcR and Gl-CalpT was highly correlated, suggesting that CalpT is directly regulated by the ecdysone receptor. cGMP is a second messenger inhibiting ecdysteroidogesis in the molting gland (Y-organ) by molt-inhibiting hormone (MIH), suggesting that MIH action is mediated by a nitric oxide (NO)-sensitive guanylyl cyclase. The potential role of a NO signal transduction pathway was investigated. Using various PCR techniques, a full-length cDNA encoding land crab NO synthase (NOS) was isolated. The Gl-NOS cDNA encoded a protein containing 1199 amino acids with an estimated mass of ~136 kDa. RT-PCR showed that NOS was expressed in Y-organ, eyestalk ganglion, thoracic ganglion, ovary, testis, and gill. This is the first report of NOS in non-neuronal tissues, which suggests that NOS is involved in regulating ecdysteroid metabolism in the Y-organ and other tissues.