Peptides (v.80, #C)

Introduction by Ronald J. Nachman (1-3).

Kbot55, purified from Buthus occitanus tunetanus venom, represents the first member of a novel α-KTx subfamily by Rym ElFessi-Magouri; Steve Peigneur; Oussema Khamessi; Najet Srairi-Abid; Mohamed ElAyeb; Bea Garcia Mille; Eva Cuypers; Jan Tytgat; Riadh Kharrat (4-8).
Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.
Keywords: Scorpion potassium toxins; α-KTx31 subfamily; Peptide; Scorpion envenomation; Disulfide bridges;

Isolation and characterization of Ts19 Fragment II, a new long-chain potassium channel toxin from Tityus serrulatus venom by Felipe Augusto Cerni; Manuela Berto Pucca; Fernanda Gobbi Amorim; Karla de Castro Figueiredo Bordon; Julien Echterbille; Loïc Quinton; Edwin De Pauw; Steve Peigneur; Jan Tytgat; Eliane Candiani Arantes (9-17).
Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is a protein presenting 49 amino acid residues, three disulfide bridges, M r 5534 Da and was classified as a new member of class (subfamily) 2 of the β-KTxs, the second one described for Ts scorpion. The β-KTx family is composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and a C-terminal CSαβ domain (CCD), with Kv blocking activity. The extensive electrophysiological screening (16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blocking effect on Kv1.2 (IC50 value of 544 ± 32 nM). However, no cytolytic activity was observed with this toxin. We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19 Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesized the peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed to be cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and its fragments (I–III) are also discussed here. A mechanism of post-translational processing (post-splitting) is suggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this report provides further evidence for the existence of several compounds in the scorpion venom contributing to the diversity of the venom arsenal.
Keywords: Tityus serrulatus; Scorpion toxin; Scorpion β-KTxs; Electrophysiology; Potassium channels; Post-splitting;

[des-Arg1]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom by Bruno Duzzi; Daniela Cajado-Carvalho; Alexandre Kazuo Kuniyoshi; Roberto Tadashi Kodama; Fabio Cesar Gozzo; Mariana Fioramonte; Denise Vilarinho Tambourgi; Fernanda Vieira Portaro; Vanessa Rioli (18-24).
The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg1]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg1]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg1]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg1]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg1]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.
Keywords: Tityus serrulatus; Neprilysin (NEP); Proctolin; [des-Arg1]-proctolin; Cockroach; Scorpion venom; Neuropeptide;

A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)6W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10−10–10−5  M.
Keywords: Allatostatin-B; Ca2+ imaging; Gryllus bimaculatus; Receptor;

Characterisation of two conopressin precursor isoforms in the land snail, Theba pisana by M.J. Stewart; B.I. Harding; K.J. Adamson; T. Wang; K.B. Storey; S.F. Cummins (32-39).
Increased understanding of the molecular components involved in mollusc reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring. The neuropeptide conopressin, a member of the vasopressin/oxytocin-like peptide family, can modulate various reproductive activities in invertebrates. In this study, we used the hermaphroditic land snail, Theba pisana, to investigate the presence and tissue-specific distribution of a conopressin gene. Our transcriptomic analysis of T. pisana CNS sheath tissue has revealed two conopressin gene transcripts (Tpi-conopressin-1 and Tpi-conopressin-2), each encoding for precursors containing an identical conopressin nonapeptide and a variable neurophysin. T. pisana conopressins share high identity with other land snails and slugs, as well as other mollusc and vertebrate vasopressin/oxytocin, supported by phylogenetic analysis. Conserved residues in the T. pisana neurophysin are important for peptide binding, and we present molecular dynamic models demonstrating the most likely stable structure of the Tpi-conopressin-1 peptide when associated with neurophysin. RT-PCR shows that Tpi-conopressin-1 is additionally expressed in reproductive tissues, including the dart sac, where abundant spatial expression throughout the sac region is found; this implies a role in ‘love’ dart synthesis or dart injection during mating. The presence of a conopressin receptor in the CNS sheath indicates CNS neural excitation. In summary, this study represents a detailed molecular analysis of conopressin in a land snail.
Keywords: Conopressin; Neurophysin; CNS; Dart sac; Theba pisana; Land snail;

Molecular cloning, expression analysis and cellular localization of an LFRFamide gene in the cuttlefish Sepiella japonica by Zi-hao Cao; Lian-lian Sun; Chang-feng Chi; Hui-hui Liu; Li-qing Zhou; Zhen-ming Lv; Chang-wen Wu (40-47).
Neuropeptides are important regulators of physiological processes in metazoans, such as feeding, reproduction, and heart activities. In this study, an LFRFamide gene was identified from the cuttlefish Sepiella japonica (designated as SjLFRFamide). The full-length sequence of SjLFRFamide cDNA has 841 bp, and the open reading frame contains 567 bp encoding 188 amino acids, which shared high similarity with precursor SOFaRP2 from Sepia officinalis. The deduced SjLFRFamdie precursor protein contains a signal peptide and four different FLPs (FMRFamide-like peptides): one pentapeptide (TIFRFamide), two hexapeptides (NSLFRFamide and GNLFRFamide) and one heptapeptide (PHTPFRFamide). Multiple sequence alignment showed that SjLFRFamide contains rather conserved mature peptides, which all ended in FRF. The phylogenetic analysis suggests that SjLFRFamide belongs to the LFRFamide subfamily. The tissue distribution analysis through quantitative real-time PCR method showed that SjLFRFamide mRNA is significantly expressed in the brain, and slight trace are detected in female nidamental gland and accessory nidamental gland. In situ hybridization assay of the brain indicated that SjLFRFamide is transcribed in several different functional lobes, suggesting SjLFRFamide might associate with multiple physiological regulations, such as feeding, chromatophore regulation and reproduction. This is the first study describing LFRFamide in S. japonica, which might have great importance for cuttlefish artificial breeding.
Keywords: Neuropeptide; FMRFamide-like peptides; LFRFamide; Cuttlefish; Sepiella japonica;

Display OmittedIn the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91 Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645 bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.
Keywords: Bactrocera dorsalis; Male accessory glands; Transcriptome; Immunity; Antimicrobial peptide; Reproduction;

Differential peptide expression in the central nervous system of the land snail Theba pisana, between active and aestivated by K.J. Adamson; T. Wang; B.A. Rotgans; A.V. Kuballa; K.B. Storey; S.F. Cummins (61-71).
Hypometabolism is a physiological state of dormancy entered by many animals in times of environmental stress. There are gaps in our understanding of the molecular components used by animals to achieve this metabolic state. The availability of genomic and transcriptome data can be useful to study the process of hypometabolism at the molecular level. In this study, we use the land snail Theba pisana to identify peptides that may be involved in the hypometabolic state known as aestivation. We found a total of 22 neuropeptides in the central nervous system (CNS) that were differentially produced during activity and aestivation based on mass spectral-based neuropeptidome analysis. Of these, 4 were upregulated in active animals and 18 were upregulated in aestivation. A neuropeptide known to regulate muscle contractions in a variety of molluscs, the small cardioactive peptide A (sCAPA), and a peptide of yet unknown function (termed Aestivation Associated Peptide 12) were chosen for further investigation using temporal and spatial expression analysis of the precursor gene and peptide. Both peptides share expression within regions of the CNS cerebral ganglia and suboesophageal ganglia. Relative transcript abundance suggests that regulation of peptide synthesis and secretion is post-transcriptional. In summary, we provide new insights into the molecular basis of the regulation of aestivation in land snails through CNS peptide control.
Keywords: Snail; Theba pisana; Peptides; Neuropeptides; Aestivation; sCAP; Central nervous system;

Expression of RYamide in the nervous and endocrine system of Bombyx mori by Ladislav Roller; Daniel Čižmár; Branislav Bednár; Dušan Žitňan (72-79).
RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12–14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion.
Keywords: Neuropeptide; Neuron; Enteroendocrine cell; Immunohistochemistry; In situ hybridization;

An overview of antifungal peptides derived from insect by Mohammad Omer Faruck; Faridah Yusof; Silvia Chowdhury (80-88).
Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.
Keywords: Amino acid; Protein; Fungi; Antifungal peptides; Insect;

Display OmittedInsects obtain energy and nutrients via feeding to support growth and development. The insulin signaling pathway is involved in the regulation of feeding; however, the underlying mechanisms are not fully understood. Here, we show that insulin signaling regulates food intake via crosstalk with neuropeptide sulfakinin in the red flour beetle, Tribolium castaneum. Silencing of the insulin receptor (InR) decreased the food intake in the penultimate and final instar stages, leading to a decrease of weight gain and mortality during larval-pupal metamorphosis. Interestingly, the knockdown of InR co-occurred with an increased expression of sulfakinin (sk), a gene encoding neuropeptide SK functioning as a satiety signal. In parallel, double silencing of sk and InR eliminated the inhibitory effect on food intake as induced by silencing of InR and the larvae died as prepupae. In conclusion, this study shows, for the first time, that the insulin/InR signaling regulates food intake through the sulfakinin signaling pathway in the larval stages of this important model and pest insect, indicating a novel target for pest control.
Keywords: Insulin receptor; Food intake; Sulfakinin; RNA interference; Tribolium castaneum;

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster by Elizabeth Cannell; Anthony J. Dornan; Kenneth A. Halberg; Selim Terhzaz; Julian A.T. Dow; Shireen-A. Davies (96-107).
Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly.Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways.Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.
Keywords: DH44; Kinin; Desiccation; Starvation; Neuropeptide receptor; Drosophila melanogaster;

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.
Keywords: Diuresis; Blood-feeding; Molting; Neuropeptide; Kinin; Peptide;