Proteomic and molecular biological studies on components of the MIH signaling pathway in the crustacean molting gland from land crab, Gecarcinus lateralis

Regulation of the molting cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland (XO/SG) complex and the Y-organ (YO). Molt-inhibiting hormone (MIH), is produced in XO/SG complex, and regulates ecdysteroidogenesis in the YO. The goal of this study was to characterize components of the MIH signaling pathway. The eyestalk is the main endocrine system to produce MIH in crustaceans, and eyestalk ablation (ESA) causes significant increases in hemolymph ecdysteroid of land crabs. However, MIH immunoprecipitation of YO extracts showed that the MIH or MIH-like peptide(s) was/were not decreased even after 3 days post ESA. Further analysis of the MIH-immunoprecipitated samples by two-dimensional gel electrophoresis showed the presence of putative MIHBPs (MIH-binding proteins) in the YOs of intact animals. This suggests that MIHBP(s) may facilitate the binding of MIH to receptor, which would result in the suppression of ecdysteroidogenesis. MIHBPs were shown at 35 kDa, pI 7.6 and 32 kDa, pI 8.0. These proteins remain to be identified, but preliminary sequences obtained by tandem mass spectrometry showed they are related. MIH induces at least 60-fold increase in cGMP in the YO. Three guanylyl cyclases (GCs) were cloned from land crabs and each GC expression was investigated after ESA to identify which GC is regulated in the Y-organ. RT-PCR revealed that expression of both NO-sensitive (Gl-GC-Ibeta) and NO-insensitive soluble (Gl-GC-III) GCs increased in response to elevated ecdysteroid induced by ESA. One of the, receptor guanylyl cyclase isoforms found in this study, Gl-GC-II(+18), was expressed predominantly in striated muscle. Semi-quantitative PCR and statistical analysis showed that the expressional change of this isoform was significant only in claw muscle in response to elevated ecdysteroid induced by ESA. The response of Gl-GC-II(+18) to elevated ecdysteroid in claw muscle indicates that a specific ligand binds to this receptor and triggers a series of signaling cascades for claw muscle atrophy, eventually inducing molting. Total protein extracts of Y-organs from intact and ES ablated animals were analyzed to discover potential candidates of MIH signaling by two-dimensional gel electrophoresis, image analysis, and proteome clustering. As a result, more than 3-fold increases in 170 proteins and 3-fold decreases in 89 proteins out of total 543 proteins were identified, suggesting that expression proteomics has a powerful potential as high-throughput target screening system. Phosphoproteins purified from Y-organs were analyzed by two-dimensional gel electrophoresis. ESA caused significant changes in the levels of phosphoproteins. These proteins were selected as putative candidates for molt-regulating factors. One of these phosphoproteins was detected on a Western blot of ESA Y-organ extracts probed with an anti-NOS antibody. Peptide mass fingerprinting analysis confirmed that the protein was land crab NOS. These results indicate that NOS phosphorylation is associated with YO activation in ESA animals. A MIH signaling pathway involving NOS and NO-sensitive GC is proposed.