Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

Possible role of non-classical chromatophorotropins on the regulation of the crustacean erythrophore.

Two neuropeptides, the pigment dispersing hormone (PDH) and the pigment concentrating hormone (PCH), are well known to respectively promote centrifugal and centripetal granule translocation in the freshwater shrimp Macrobrachium potiuna erythrophores. Herein, we demonstrate for the first time the effects of crustacean non-classical chromatophorotropins on the pigment migration in M. potiuna erythrophores. Although proctolin, 20-hydroxyecdisone (20HE), and melatonin were ineffective, the crustacean cardioactive peptide (CCAP) was a full agonist, inducing pigment dispersion in a dose-dependent manner with EC(50) of 9.5. 10(-7) M. In addition, concentrations of CCAP lower than the minimal effective dose (10(-8) and 10(-7) M) decreased the PCH-induced aggregation, shifting rightward the dose-response curve (DRC) to PCH 2.2- and 29-fold, respectively. Surprisingly, melatonin (10(-7) and 10(-6) M) also shifted to the right 8.7- and 46.5-fold, respectively, the DRC to PCH. In conclusion, our data demonstrate that besides PCH and PDH, CCAP and melatonin also regulate the pigment migration within the crustacean erythrophore. J. Exp. Zool. 284:711-716, 1999.Copyright 1999 Wiley-Liss, Inc.     

Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and protein hormones

We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala(7)-CCAP, CCHamide, Arg(7)-corazonin, DENamides, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin, two alternative splice forms of ion transport peptide (ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met(4)-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, and three tachykinins. There are two genes for a preprohormone containing orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.     

A structural and functional comparison of nematode and crustacean PDH-like sequences

The elucidation of the whole genome of the nematode Caenorhabditis elegans allowed for the identification of ortholog genes belonging to the pigment dispersing hormone/factor (PDH/PDF) peptide family. Members of this peptide family are known from crustaceans, insects and nematodes and seem to exist exclusively in ecdysozoans where they play a role in different processes, ranging from the dispersion of integumental and eye (retinal) pigments in decapod crustaceans to circadian rhythms in insects and locomotion in C. elegans. Two pdf genes (pdf-1 and pdf-2) encoding three different peptides: PDF-1a, PDF-1b and PDF-2 have been identified in C. elegans. These three C. elegans PDH-like peptides are similar but not identical in primary structure to PDHs from decapod crustaceans. We investigate whether this divergence has an influence on the pigment dispersing function of the peptides in a decapod crustacean, namely the shrimp Palaemon pacificus. We show that C. elegans PDF-1a and b peptides display cross-functional activity by dispersing pigments in the epithelium of P. pacificus at physiological doses. Moreover, by means of a comparative amino acid sequence analysis of nematode and crustacean PDH-like peptides, we can pinpoint several potentially important residues for eliciting pigment dispersing activity in decapod crustaceans. Although there is no sequence information on a receptor for PDH in decapod crustaceans, we postulate that there is general conservation of the PDH/PDF signaling system based on structural similarities of precursor proteins and receptors (including those from a branchiopod crustacean and from C. elegans).     

New insights into evolution of crustacean hyperglycaemic hormone in decapods--first characterization in Anomura

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.     

Circadian rhythm of pigment migration induced by chromatrophorotropins in melanophores of the crab Chasmagnathus granulata

The circadian rhythm of black pigment migration of melanophores of the crab Chasmagnathus granulata and the variation in responsiveness of these cells to pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide (CCAP), and red pigment-concentrating hormone (RPCH) were investigated. Melanophores of C. granulata possess an endogenous circadian rhythm of pigment migration, with black pigments staying more dispersed during the day period and more aggregated during the night period. This rhythm seems to be largely dependent on an endogenous release of neurohormones from eyestalks, and to a lesser extent on a primary response to illumination. beta-PDH was the most potent PDH isoform to induce pigment dispersion in both in vivo (EC50 = 0.4 pmol/animal) and in vitro (EC50 = 0.18 microM) assays. CCAP also induced pigment dispersion in vivo and in vitro assays (EC50 = 12 microM), but it was less potent than beta-PDH. In vivo, RPCH induced a low and nondose-dependent pigment aggregation, while in vitro, it had no effect on pigment migration. The responsiveness of melanophores of C. granulata to beta-PDH was significantly higher during the day period when compared to the night period in both assays, in vitro and in vivo. These results suggest that the endogenous circadian rhythm of black pigment migration is dependent on both endogenous circadian rhythm of beta-PDH synthesis and/or release from eyestalks and on an endogenous rhythm of responsiveness of melanophores to beta-PDH.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.     

Molecular biology of neurohormone precursors in the eyestalk of Crustacea

Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.     

Quantitative in vitro assay for crustacean chromatophorotropins and other pigment cell agonists

An in vitro crustacean (freshwater shrimp, Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 x 10(-12) M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the ionophore-induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore-treated erythrophores. The potencies of alpha-PDH and beta-PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure-activity studies of the various chromatophorotropins and their analogs.     

Proteomics and signal transduction in the crustacean molting gland

Regulation of the molting cycle in decapod crustaceans involves2 endocrine organs: the X-organ/sinus gland (XO/SG) complexlocated in the eyestalk ganglia and the Y-organ (YO) locatedin the cephalothorax. Two neuropeptides [molt-inhibiting hormone(MIH) and crustacean hyperglycemic hormone (CHH)] are producedin the XO/SG complex and inhibit ecdysteroidogenesis in theYO. 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 neuropeptidesuppression of the YO. Proteomics was used to identify potentialcomponents of signal transduction pathways ("targeted" or cell-mapproteomics) as well as assess the magnitude of protein changesin response to activation ("global" or expression proteomics)in the tropical land crab, Gecarcinus lateralis. Total proteinsin YOs from intact and ES-ablated animals were separated bytwo-dimensional gel electrophoresis and expression profileswere assessed by image analysis and gene clustering software.ESA caused a >3-fold increase in the levels of 170 proteinsand >3-fold decrease in the levels of 89 proteins; a totalof 543 proteins were quantified in total YO extracts. ESA inducedsignificant changes in the levels of 3 groups of proteins elutingfrom a phosphoprotein column and detected with phosphoproteinstaining of two-dimensional gels;     

Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.     

Chemical Properties and Physiological Actions of Crustacean Chromatophorotropins

The crustacean pigment-translocating hormones, the red pigment-concentratinghormone (RPCH), an octapeptide, and the light-adapting distalretinal pigment hormone (DRPH), an octadecapeptide, are thefirst invertebrate neurohormones to be fully characterized.Studies with both purified and synthetic hormones show that,in certain decapods, RPCH is a general pigment-concentratinghormone (PCH), affecting the pigments of all kinds of chromatophores(erythrophores, xanthophores, leucophores and melanophores);the DRPH seems to serve not only light-adapting function, butalso act as a general chromatophore pigment-dispersing hormone(PDH). The two hormones thus function as antagonists when regulatingthe color-adaptation of the decapod crustaceans. PCH activityis widely distributed within the arthropod endocrine systems.The first characterized insect neurohormones, the locust adipokinetichormones (AKH I and AKH II), show close structural similaritiesto the crustacean hormone, indicating a common evolution ofsome of the arthropod neurohormones. Physiological studies ofthe three hormones (RPCH, AKH I, and AKH II) and their syntheticanalogs show that they crossreact, i.e., they all exhibit pigment-concentratingactivity when tested on decapod crustaceans, adipokinetic activitywhen tested on locusts, and hyperglycemic activity when testedon cockroaches, although each of the hormones is more potentin its own system. Structure-function studies show, however,that quite different binding-site requirements exist for thehormones in activating their receptors on the various targettissues. The physiological specificity in their action thereforeseems to depend on a differential evolution of the hormone receptors.