Proteomics and signal transduction in the crustacean molting gland

Abstract

Regulation of the molting cycle in decapod crustaceans involves 2 endocrine organs: the X-organ/sinus gland (XO/SG) complex located in the eyestalk ganglia and the Y-organ (YO) located in the cephalothorax. Two neuropeptides [molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH)] are produced in the XO/SG complex and inhibit ecdysteroidogenesis in the YO. Thus, YO activation is induced by eyestalk ablation (ESA), which removes the primary source of MIH and CHH. Cyclic nucleotides (cAMP and cGMP) and nitric oxide (NO) appear to mediate neuropeptide suppression of the YO. Proteomics was used to identify potential components of signal transduction pathways ("targeted" or cell-map proteomics) as well as assess the magnitude of protein changes in response to activation ("global" or expression proteomics) in the tropical land crab, Gecarcinus lateralis. Total proteins in YOs from intact and ES-ablated animals were separated by two-dimensional gel electrophoresis and expression profiles were assessed by image analysis and gene clustering software. ESA caused a >3-fold increase in the levels of 170 proteins and >3-fold decrease in the levels of 89 proteins; a total of 543 proteins were quantified in total YO extracts. ESA induced significant changes in the levels of 3 groups of proteins eluting from a phosphoprotein column and detected with phosphoprotein staining of two-dimensional gels; ∼17 kDa and ∼150 kDa phosphoproteins increased in activated YOs, while ∼12 kDa phosphoproteins decreased. A ∼150 kDa phosphoprotein, which was isolated only from activated YO, was identified as NO synthase by western blotting and mass spectrometry of trypsin peptides. These data show that phosphorylation of NO synthase is associated with activation of the YO. A neuropeptide signaling pathway involving NO synthase and NO-sensitive guanylyl cyclase is proposed.