Three calpains and ecdysone receptor in the land crabGecarcinus 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.

Ecdysone receptor in the mud crab Scylla paramamosain: a possible role in promoting ovarian development

“…In crustaceans, it is generally known that ecdysteroids are first synthesized in the Y-organ (molting gland), and subsequently released into hemolymph where they are hydroxylated to become 20-hydroxyecdysone (20E), a biologically active formwhich belongs to the nuclear receptor family, to activate DNA regulatory element (Bortolin et al 2011, Gaertner et al 2012. In crustaceans, EcR can form a heterodimer with another nuclear receptor known as retinoid X receptor (RXR), which is orthologous with ultraspiracle (USP) in insects, to regulate the downstream genes in the ecdysteroid signaling pathway (Wu et al 2004, Kim et al 2005a,b, Hopkins et al 2008, Hill et al 2013.…”

Endocrine regulation of the reproduction in crustaceans: Identification of potential targets for toxicants and environmental contaminants

“…Recent studies with U. pugilator also suggested temporal-and organ/tissue-specific distribution of several EcR isoforms (Durica et al 2002). It may be deduced that various EcRs control specific responses in different organs and determine thus tissue sensitivity to ecdysteroids (Kim et al 2005a;Zou 2005).…”

“…EcR belongs to a nuclear receptor family (receptors acting as transcription factors), and it has a typical gene composition of five domains from which DNA-binding domain (DBD) and ligand-binding domain (LBD) are phylogenetically conserved. Analyses of these two domains in Uca pugilator (Durica et al 2002) and Gecarcinus lateralis Freminville, 1835 (Kim et al 2005a) revealed high similarity to EcR from insects. Recent studies with U. pugilator also suggested temporal-and organ/tissue-specific distribution of several EcR isoforms (Durica et al 2002).…”

“…Several of such studies in crustaceans add new information to mechanisms and pathways previously described only in traditional insect and/or vertebrate models (Verslycke et al 2002;Wu et al 2004;Kim et al 2005a). Fig.…”

Parasites, proteomics and performance: effects of gregarine gut parasites on dragonfly flight muscle composition and function

“…Kim et al, 2005). Pemrick and Grebenau reported that proteolytic activity by calcium-activated neutral protease (CANP; also known as calpain) in the head region of ~200·kDa MHC from rabbit muscle produced 180, 165 and 150·kDa sized MHC peptides (Pemrick and Grebenau, 1984).…”