A proteomic approach to neuropeptide function elucidation

Many of the diverse functions of neuropeptides are still elusive. As they are ideally suited to modulate traditional signaling, their added actions are not always detectable under standard laboratory conditions. The search for function assignment to peptide encoding genes can therefore greatly benefit from molecular information. Specific molecular changes resulting from neuropeptide signaling may direct researchers to yet unknown processes or conditions, for which studying these signaling systems may eventually lead to phenotypic confirmation. Here, we applied gel-based proteomics after pdf-1 neuropeptide gene knockout in the model organism Caenorhabditis elegans. It has previously been described that pdf-1 null mutants display a locomotion defect, being slower and making more turns and reversals than wild type worms. The vertebrate functional homolog of PDF-1, vasocative intestinal peptide (VIP), is known to influence a plethora of processes, which have so far not been investigated for pdf-1. Because proteins represent the actual effectors inside an organism, proteomic analysis can guide our view to novel pdf-1 actions in the nematode worm. Our data show that knocking out pdf-1 results in alteration of levels of proteins involved in fat metabolism, stress resistance and development. This indicates a possible conservation of VIP-like actions for pdf-1 in C. elegans.     

Crustacean Pigmentary-Effector Hormones: Chemistry and Functions of RPCH, PDH, and Related Peptides

Integumental color changes and eye pigment movements in crustaceansare regulated by pigmentary-effector hormones. The identifiedhormones include: an octapeptide RPCH (red pigment-concentratinghormone) and several forms of octadecapeptide PDH (pigment-dispersinghormone: -PDH, ß-PDH). RPCH-related peptides (AKHs,adipokinetic hormones) and PDH-related peptides (PDFs, pigment-dispersingfactors) occur in insects, and are recognized as members ofAKH/RPCH and PDH/PDF peptide families. The domain for maturepeptide is located between the signal peptide and precursor-relatedpeptide in AKH/RPCH precursors, and at the C-terminal end inthe PDH/PDF precursors. The precursor-related (associated) peptidesin RPCH and PDH precursors in Crustacea show little or no similarityto corresponding domains of AKH and PDF precursors in insects.Although the functions of precursor-related peptides are unknown,the mature peptides are shown to serve diverse functions. RPCH'sactions in crustaceans include: pigment concentration in oneor more types of chromatophores, dark-adaptational screeningpigment movement in distal eye pigment cells, increase of retinalsensitivity, and neuromodulation. The related AKHs largely influencemetabolism in insects, although they serve additional functions.PDHs trigger pigment dispersion in chromatophores and inducelight-adaptational screening pigment movements in extraretinulareye pigment cells. The related PDFs appear to serve as a transmitterof circadian signals in the regulation of biological rhythmsin insects. Evolutionary relationships among the PDH/PDF peptidesand directions for future research are discussed.     

Pigment dispersing hormone generates a circadian response to light in the crayfish, Procambarus clarkii

Photoreceptor cells have been identified as important structures in the organization of the circadian system responsible for the generation and expression of the electroretinogram (ERG) circadian rhythm. They are the structures where the circadian periodicity is expressed (effectors) and which transform information from external light signals to be conducted to the pacemaker in order to induce adjustments of the rhythm (synchronizers). After isolation, eyestalks perfused in a pigment dispersing hormone (PDH) solution, show significant changes in receptor potential (RP) amplitude and duration. Exogenous PDH injected into intact crayfish induces a migration of retinal shielding pigments to a light-adapted state. A single dose of PDH produces advances or delays in the circadian rhythm of response to light of visual photoreceptors. All these effects depend on the circadian phase of PDH application. Consequently, the determination of the action of exogenous PDH on photoreceptor cells proved to be very helpful in understanding some mechanisms underlying the circadian organization of crayfish.     

Functional characterization of three G protein-coupled receptors for pigment dispersing factors in Caenorhabditis elegans

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.     

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.     

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.     

sem-4/spalt and egl-17/FGF have a conserved role in sex myoblast specification and migration in P. pacificus and C. elegans

Evolutionary comparisons between Caenorhabditis elegans and the satellite organism Pristionchus pacificus revealed major differences in the regulation of nematode vulva development. For example, Wnt signaling is part of a negative signaling system that prevents vulva formation in P. pacificus, whereas it plays a positive role in C. elegans. We wondered if the genetic control of the second major part of the nematode egg-laying system, the sex muscles, has diverged similarly between P. pacificus and C. elegans. The sex muscles derive from the mesoblast M, which has an identical lineage in both species. Here, we describe a large-scale mutagenesis screen for mutations that disrupt the M lineage and the sex myoblast (SM) sublineage. We isolated and characterized mutations that result in a failure of proper SM fate specification and SM migration and showed that the corresponding genes encode Ppa-sem-4 and Ppa-egl-17, respectively. Ppa-sem-4 mutants have additional defects in the specification of the vulva precursor cells P(5, 7).p and experimental studies in the Ppa-egl-17 mutant background indicate a complex set of gonad-dependent and gonad-independent mechanisms required for SM migration. Mutations in Cel-sem-4 and Cel-egl-17 cause similar defects. Thus, the molecular mechanisms of SM cell specification and migration are conserved between P. pacificus and C. elegans.     

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques

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. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.     

Pigment dispersing hormone modulates spontaneous electrical activity of the cerebroid ganglion and synchronizes electroretinogram circadian rhythm in crayfish Procambarus clarkii

In crayfish, one very well-studied circadian rhythm is that of electroretinogram (ERG) amplitude. The cerebroid ganglion has been considered a plausible site for the circadian pacemaker of this rhythm and for the retinular photoreceptors, as the corresponding effectors. The pigment dispersing hormone (PDH) appears to synchronize ERG rhythm, but its characterization as a synchronizer cue remains incomplete. The main purposes of this work were a) to determine whether PDH acts on the cerebroid ganglion, and b) to complete its characterization as a non-photic synchronizer. Here we show that PDH increases the number of the spontaneous potentials of the cerebroid ganglion, reaching 149.92 ± 6.42% of the activity recorded in the controls, and that daily application of PDH for 15 consecutive days adjusts the ERG circadian rhythm period to 24.0 ± 0.2 h and the end of the activity period of the rhythm coincides with the injection of the hormone. In this work, we hypothesized that in crayfish, PDH transmits the “day” signal to the ERG circadian system and acts upon both the presumptive circadian pacemaker and the corresponding effectors to reinforce the synchronization of the system.     

Differential distribution of β-pigment-dispersing hormone (β-PDH)-like immunoreactivity in the stomatogastric nervous system of five species of decapod crustaceans

Summary Pigment-dispersing hormone (PDH) acts to disperse pigments within the chromatophores of crustaceans. Using an antibody raised against -PDH from the fiddler crab Uca pugilator, we characterized the distribution of -PDH-like immunoreactivity in the stomatogastric nervous system of five decapod crustaceans: the crabs, Cancer borealis and Cancer antennarius, the lobsters, Panulirus interruptus and Homarus americanus, and the crayfish, Procambarus clarkii. No somata were stained in the stomatogastric ganglion (STG) or the esophageal ganglion in any of these species. Intense PDH-like staining was seen in the neuropil of the STG in P. interruptus only. In all 5 species, cell bodies, processes, and neuropil within the paired circumesophageal ganglia (CGs) showed PDH-like staining; the pattern of this staining was unique for each species. In each CG, the -PDH antibody stained: 1 large cell in C. borealis; 3 small to large cells in C. antennarius; 3–8 medium cells in P. clarkii; 1–4 small cells in H. americanus; and 13–17 small cells in P. interruptus. The smallest cell in each CG in C. antennarius sends its axon, via the inferior esophageal nerves, into the opposite CG; this pair of cells, not labeled in the other species studied, may act as bilateral coordinators of sensory or motor function. These diverse staining patterns imply some degree of evolutionary diversity among these crustaceans. A -PDH-like peptide may act as a neuromodulator of the rhythms produced by the stomatogastric nervous system of decapod crustaceans.     

A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus

To understand the evolution of developmental processes, nonmodel organisms in the nematodes, insects, and vertebrates are compared with established model systems. Often, these comparisons suffer from the inability to apply sophisticated technologies to these nonmodel species. In the nematode Pristionchus pacificus, cellular and genetic analyses are used to compare vulva development to that of Caenorhabditis elegans. However, substantial changes in gene function between P. pacificus and C. elegans limit the use of candidate gene approaches in studying P. pacificus mutations. To facilitate map-based cloning of mutations in P. pacificus, we constructed a BAC-based genetic linkage map. A BAC library of 13,440 clones was generated and completely end sequenced. By comparing BAC end and EST sequences between the "wild-type" strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington, 133 single-stranded conformational polymorphisms were identified. These markers were tested on a meiotic mapping panel of 46 randomly picked F(2) animals after a cross of the two strains, providing the first genetic linkage map of P. pacificus. A mapping strategy using two selected markers per chromosome was devised and the efficiency of this approach was illustrated by the mapping of the Ppa-unc-1/Twitchin gene.     

The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens

Bacterial pathogens have shaped the evolution and survival of organisms throughout history, but little is known about the evolution of virulence mechanisms and the counteracting defence strategies of host species. The nematode model organisms, Caenorhabditis elegans and Pristionchus pacificus, feed on a wealth of bacteria in their natural soil environment, some of which can cause mortality. Previously, we have shown that these nematodes differ in their susceptibility to a range of human and insect pathogenic bacteria, with P. pacificus showing extreme resistance compared with C. elegans. Here, we isolated 400 strains of Bacillus from soil samples and fed their spores to both nematodes. Spores of six Bacillus strains were found to kill C. elegans but not P. pacificus. While the majority of Bacillus strains are benign to nematodes, observed pathogenicity is restricted to either the spore or the vegetative stage. We used the rapid C. elegans killer strain (Bacillus sp. 142) to conduct a screen for hypersusceptible P. pacificus mutants. Two P. pacificus mutants with severe muscle defects and an extended defecation cycle that die rapidly on Bacillus spores were isolated. These genes were identified to be homologous to C. elegans, unc-22 and unc-13. To test whether a similar relationship between defecation and bacterial pathogenesis exists in C. elegans, we used five known defecation mutants. Quantification of the defecation cycle in mutants also revealed a severe effect on survival in C. elegans. Thus, intestinal peristalsis is critical to nematode health and contributes significantly to survival when fed Gram-positive bacteria.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.     

Comparative genetics: a third model nematode species

Recent studies have introduced Oscheius sp. CEW1 as a third nematode species accessible to genetic analysis, joining the better known Caenorhabditis elegans and Pristionchus pacificus. A group of vulva-defective mutants in Oscheius has been identified, with defects not seen in C. elegans.     

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.