Elke Clynen

Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC‐2/EGL‐3): mutant analysis by mass spectrometry

Biologically active peptides are synthesized as larger inactive proprotein peptide precursors which are processed by the concerted action of a cascade of enzymes. Among the proprotein convertases, PC2 is widely expressed in neuro‐endocrine tissues and has been proposed to be the major convertase involved in the biosynthesis of neuropeptides. In this study, we have examined the role of the Caenorhabditis elegans orthologue PC2/EGL‐3 in the processing of proprotein peptide precursors. We recently isolated and identified 60 endogenous peptides in the nematode C. elegans by two‐dimensional nanoscale liquid chromatography – quadrupole time‐of‐flight tandem mass spectrometry. In the present study, we compare the peptide profile of different C. elegans strains, including PC2/EGL‐3 mutants. For this purpose, we used an offline approach in which HPLC fractions are analysed by a matrix‐assisted laser desorption ionisation – time of flight mass spectrometer. This differential peptidomic approach unambiguously provides evidence for the role of PC2/EGL‐3 in the processing of FMRFamide‐like peptide (FLP) precursors and neuropeptide‐like protein (NLP) precursors in nematodes.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

Peptidomic survey of the locust neuroendocrine system.

Neuropeptides are important controlling agents in animal physiology. In order to understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. We here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, we analysed the brain, suboesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e. pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosaemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.

The use of peptidomics in endocrine research

In 2002, the Nobel Prize for chemistry was awarded to the inventors of two novel ionization techniques in mass spectrometry, MALDI and ESI. These techniques, often in combination with data from genomic databases, represent an extremely powerful tool in analytical (bio)chemistry, with many applications, e.g., in the field of proteomics. Peptides, which are small proteins, have, despite their importance as controlling agents in numerous physiological processes, as yet been much less intensively studied by these novel techniques than larger proteins. The term peptidomics, i.e., the study of all peptides expressed by a certain cell, organ or organism was only introduced in 2001. In neuroendocrinology, spectacular progress could already be realized and the future looks bright. In this minireview we discuss the different methodologies that are used in peptidomics and give an overview of the wide range of applications.

Annotation of novel neuropeptide precursors in the migratory locust based on transcript screening of a public EST database and mass spectrometry

BackgroundFor holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information.ResultsRecently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques.ConclusionIn this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

Mass spectrometric analysis of the perisympathetic organs in locusts: identification of novel periviscerokinins.

A mass spectrometric analysis carried out to determine the peptidome of the abdominal perisympathetic organs in the locust species Locusta migratoria and Schistocerca gregaria yielded a number of predominant ion peaks, among which are Lom-PVK (AAGLFQFPRVamide) and Scg-MT-2 (TSSLFPHPRLamide). In addition, three novel peptides were identified: Lom-PVK-2 (identical in Schistocerca): GLLAFPRVamide, Lom-PVK-3: DGGEPAAPLWFGPRVamide, and Scg-PVK-3: DGAETPGAAASLWFGPRVamide. An extensive mass spectrometric study of the central nervous system showed that the periviscerokinins (-PRVamides) and Scg-MT-2 (-FXXPRLamide) are restricted to the abdominal ganglia and their perisympathetic organs, while the pyrokinins (-FXPRLamides) are present only in the brain-retrocerebral complex. Sequence comparison with the Drosophila genes supports a conserved gene structure whereby a capability-like gene encodes the periviscerokinins that are expressed in the abdominal ganglia and stored in the perisympathetic organs, while a hugin-like gene encodes the pyrokinins that are expressed in the head ganglia and stored in the retrocerebral complex.

Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function.

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7–30 μg and a paralysis or lethal effect was observed at 30 μg of venom (death at 1.5 μg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 μg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage‐gated Na+ and K+ channels. Some fractions block K+ currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC‐MS/MS and MALDI‐TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.

Purification and characterization of an insulin-related peptide in the desert locust, Schistocerca gregaria: immunolocalization, cDNA cloning, transcript profiling and interaction with neuroparsin

Members of the insulin superfamily are not restricted to vertebrates, but have also been identified in invertebrate species. In the current report, we present the characterization of Scg-insulin-related peptide (IRP), an insulin-related peptide in the desert locust, Schistocerca gregaria. This peptide was isolated from corpora cardiaca (CC) extracts by means of a high-performance liquid chromatography (HPLC)-based purification strategy. Subsequent cloning and sequencing of the corresponding cDNA revealed that the encoded Scg-IRP precursor displays the structural organization that is typical for members of the insulin superfamily. Moreover, immunocytochemistry on brain tissue sections demonstrated the presence of Scg-IRP in median neurosecretory cells of the pars intercerebralis and their projections towards the storage part of the CC. Quantitative real-time RT-PCR studies revealed the presence of Scg-IRP transcripts in a variety of tissues, including nervous tissue and fat body. Furthermore, these transcripts showed a tissue- and phase-dependent, temporal regulation during the reproductive cycle of adult males and females. Finally, we demonstrated that Scg-IRP interacts in vitro with a recombinant neuroparsin, a locust protein displaying sequence similarity with vertebrate IGF binding proteins.

Neuropeptides as Targets for the Development of Anticonvulsant Drugs

Epilepsy is a common neurological disorder characterized by recurrent seizures. These seizures are due to abnormal excessive and synchronous neuronal activity in the brain caused by a disruption of the delicate balance between excitation and inhibition. Neuropeptides can contribute to such misbalance by modulating the effect of classical excitatory and inhibitory neurotransmitters. In this review, we discuss 21 different neuropeptides that have been linked to seizure disorders. These neuropeptides show an aberrant expression and/or release in animal seizure models and/or epilepsy patients. Many of these endogenous peptides, like adrenocorticotropic hormone, angiotensin, cholecystokinin, cortistatin, dynorphin, galanin, ghrelin, neuropeptide Y, neurotensin, somatostatin, and thyrotropin-releasing hormone, are able to suppress seizures in the brain. Other neuropeptides, such as arginine-vasopressine peptide, corticotropin-releasing hormone, enkephalin, β-endorphin, pituitary adenylate cyclase-activating polypeptide, and tachykinins have proconvulsive properties. For oxytocin and melanin-concentrating hormone both pro- and anticonvulsive effects have been reported, and this seems to be dose or time dependent. All these neuropeptides and their receptors are interesting targets for the development of new antiepileptic drugs. Other neuropeptides such as nesfatin-1 and vasoactive intestinal peptide have been less studied in this field; however, as nesfatin-1 levels change over the course of epilepsy, this can be considered as an interesting marker to diagnose patients who have suffered a recent epileptic seizure.

Mas-allatotropin/Lom-AG-myotropin I immunostaining in the brain of the locust, Schistocerca gregaria

Mas-allatotropin (Mas-AT) and Lom-accessory gland-myotropin I (Lom-AG-MTI) are two members of a conserved family of insect neuropeptides, collectively termed allatotropins, which have diverse functions, ranging from stimulation of juvenile hormone secretion to myotropic effects on heart and hindgut. In addition, allatotropins appear to be abundant within the nervous system, suggesting neuroactive roles. To identify neurons in the insect brain suitable for a neurophysiological analysis of the roles of allatotropins, we used antisera against Mas-AT and Lom-AG-MTI to map allatotropin-immunoreactive neurons in the brain of a suitable insect, the locust Schistocerca gregaria. Both antisera revealed basically identical staining patterns throughout the locust brain with more than 12,500 immunostained interneurons per brain hemisphere. Neurosecretory cells were not labeled, and the retrocerebral complex was devoid of immunostaining. Prominent immunoreactive cell types include about 9,600 lamina monopolar neurons, medulla to lobula interneurons, local neurons of the antennal lobe, a giant interneuron of the mushroom body, projection neurons of the glomerular lobe to the mushroom body, and three systems of tangential neurons of the central complex. Several groups of neurons showed colocalization of Mas-AT- and γ-aminobutyric acid immunostaining. Mass spectrometric analysis identified a peptide with a molecular mass identical to Lom-AG-MTI in all major parts of the locust brain but not in the retrocerebral complex. This study strongly suggests that Lom-AG-MTI is highly abundant in the locust brain, and is likely to play a neuroactive role in many brain circuits including all stages of sensory processing, learning and memory, and higher levels of motor control.