Peptides (v.41, #C)

Contents (v-vi).

CAPA-gene products in the haematophagous sandfly Phlebotomus papatasi (Scopoli) – vector for leishmaniasis disease by Reinhard Predel; Susanne Neupert; William K. Russell; Frank Hauser; David H. Russell; Andrew Li; Ronald J. Nachman (2-7).
► Sandflies are responsible for transmission of leishmaniasis and other protozoan-borne diseases in humans. ► To cope with large intake of blood meal, sandflies depend on precise regulation of water balance. ► First peptides from the tiny disease vector were identified via mass spectrometric analysis. ► CAPA-gene peptides identified are associated with diuresis in other blood-feeding insects. ► First insects which accumulate CAPA-PVKs exclusively in hormone release sites of abdominal ganglia and CAPA-PK in the corpora cardiaca.Sandflies (Phlebotominae, Nematocera, Diptera) are responsible for transmission of leishmaniasis and other protozoan-borne diseases in humans, and these insects depend on the regulation of water balance to cope with the sudden and enormous intake of blood over a very short time period. The sandfly inventory of neuropeptides, including those that regulate diuretic processes, is completely unknown. Direct MALDI-TOF/TOF mass spectrometric analysis of dissected ganglia of Phlebotomus papatasi, combined with a data-mining of sandfly genome ‘contigs’, was used to identify native CAPA-peptides, a peptide class associated with the regulation of diuresis in other hematophagous insects. The CAPA-peptides identified in this study include two CAPA-PVKs, differentially processed CAPA-PK, and an additional CAPA precursor peptide. The mass spectrometric analysis of different parts of the neuroendocrine system of the sandfly indicate that it represents the first insect which accumulates CAPA-PVKs exclusively in hormone release sites of abdominal ganglia and CAPA-PK (nearly) exclusively in the corpora cardiaca. Additionally, sandflies feature the smallest abdominal ganglia (∼35 μm) where CAPA-peptides could be detected so far. The small size of the abdominal ganglia does not appear to affect the development of the median neurosecretory system as it obviously does in another comparably small insect species, Nasonia vitripennis, in which no capa-gene expression was found. Rather, immunocytochemical analyses confirm that the general architecture in sandflies appears identical to that of much larger mosquitoes.
Keywords: Capa-gene; MALDI-TOF; Peptidomics; Insect neuropeptides; Periviscerokinins; Corazonin; Phlebotomus papatasi; Psychodidae; Differential processing;

Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis by Steve Peigneur; Annelies Van Der Haegen; Carolina Möller; Etienne Waelkens; Elia Diego-García; Frank Marí; Ryno Naudé; Jan Tytgat (8-16).
► The venoms of two cone snails, C. pictus and C. natalis, have been biochemically characterized in depth. ► Thirty-three novel peptides have been identified.Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.
Keywords: Conus pictus; Conus natalis; Cone snail; Conotoxin; A-, M-, O-superfamily;

Precursor De13.1 from Conus delessertii defines the novel G gene superfamily by Manuel B. Aguilar; Ernesto Ortiz; Quentin Kaas; Estuardo López-Vera; Baltazar Becerril; Lourival D. Possani; Edgar P. Heimer de la Cotera (17-20).
► Previously reported peptide de13a has a unique eight-Cys framework (XIII). ► The precursor (De13.1) of a de13a-related peptide (de13b) was cloned. ► Peptides de13a and de13b share high protein/nucleic acid sequence identity: 87%/90%. ► Precursor De13.1 belongs to a novel gene superfamily (superfamily G).Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatán Channel, México. This peptide has eight cysteine (Cys) residues in the unique arrangement C―C―C―CC―C―C―C, which defines the cysteine framework XIII (“―” represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.
Keywords: Conidae; Conopeptide; Conotoxin; Conus delessertii; Hydroxy-lysine; Peptide de13b;

► The adipokinetic peptides of four triatomide bugs are sequenced. ► Three novel AKHs are identified: a deca-, a nona- and an octapeptide. ► The novel synthetic AKHs cause a very mild hyperlipaemia. ► The AKH of Rhodnius prolixus is detected by mass spectrometry.Peptides of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family were isolated and sequenced from the retrocerebral corpora cardiaca of four kissing bugs which are all vectors of the protozoan Trypanosoma cruzi responsible for Chagas’ disease. The sequence of three novel AKHs were deduced from the multiple MS N electrospray mass data: the octapeptide pGlu-Leu-Thr-Phe-Ser-Thr-Asp-Trp amide (denoted Rhopr-AKH) in Rhodnius prolixus and Panstrongylus megistus, the nonapeptide pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly amide (denoted Triin-AKH) in Triatoma infestans and the decapeptide pGlu-Leu-Thr-Phe-Ser-Asp-Gly-Trp-Gly-Asn amide (denoted Dipma-AKH) in Dipetalogaster maxima. The sequences were confirmed by identical behavior of natural and synthetic forms in reversed-phase HPLC and by CID-MS mass spectra. Conspecific injections of a dose of 10 pmol of the respective synthetic peptides resulted in a small but significant increase of the lipid concentration in the hemolymph. These experiments suggest that AKHs in kissing bugs act to regulate lipid metabolism, possibly during dispersal flights which is one of the mechanisms whereby the insects reach new outbreak areas.
Keywords: Insects; Kissing bugs; Reduviidae; Adipokinetic hormone; Mass spectrometry; Lipid mobilization;

Neuropeptidomics of the Australian sheep blowfly Lucilia cuprina (Wiedemann) and related Diptera by Mohammad Mazibur Rahman; Susanne Neupert; Reinhard Predel (31-37).
► The Australian sheep blowfly Lucilia cuprina is a widespread pest species that severely affects the dry fish industry in developing countries. ► The peptidome of the CNS and the CNS-associated retrocerebral complex and abdominal perisympathetic organs was analyzed in larval and adult L. cuprina. ► The data presented give no conclusive hint for the development of peptidomimetics that would affect only Lucilia but not related flies.Insect neuropeptides are the most diverse and important group of messenger molecules that regulate almost all physiological processes, including behavior. In this study, we performed a combination of matrix assisted laser desorption ionization time of flight (MALDI-TOF) and electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry to analyze the peptidome of the brain and the neurohemal organs of the Australian sheep blowfly Lucilia cuprina and compared the data with those of related flies such as the gray flesh fly Sarcophaga (=Neobellieria) bullata; the cabbage root fly Delia radicum, the fruit fly Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti. Without counting low intensity signals of truncated peptides, 45 neuropeptides arising from 12 neuropeptide genes (adipokinetic hormone, CAPA-peptides, corazonin, extended FMRFamides, SIFamide, insect kinin, short neuropeptide F, NPLP-1 peptides, HUGIN-pyrokinin, sulfakinins, allatostatins A, putative eclosion hormone precursor peptide) were identified; sequences of extended FMRFamides were reported in a separate publication. The remarkable similarity of the peptidome of cyclorraphan flies, which contain a large number of ecologically important species, does not support the development of a species-specific neuropeptide-based insect pest control strategy. However, mass spectrometric approaches as shown here do not cover the entire peptidome or differences at the receptor level and it is possible that group-specific peptide ligands or receptors exist that escaped the detection.
Keywords: MALDI-TOF mass spectrometry; Sarcophaga; Neobellieria; capa; Periviscerokinins; Short neuropeptide F; Insect neuropeptides;

A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico by Manuel B. Aguilar; Alejandro Zugasti-Cruz; Andrés Falcón; César V.F. Batista; Baldomero M. Olivera; Edgar P. Heimer de la Cotera (38-44).
► Two six Cys-peptides (as25a and as25b) were purified from Conus cancellatus venom. ► Peptides as25a and as25b have a novel arrangement of Cys residues. ► Peptide as25a has paralytic and lethal activities. ► Peptide as25b has two hydroxyproline residues. ► Peptides as25a and as25b might represent a novel gene superfamily.The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09 Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08 Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmol (∼0.635 μg, ∼24.9 ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.
Keywords: Conus cancellatus; Conus austini; Vermivorous; Conotoxin; Six Cys; Paralysis;

Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods by Xian-Chun Zeng; Yao Nie; Xuesong Luo; Shifen Wu; Wanxia Shi; Lei Zhang; Yichen Liu; Hanjun Cao; Ye Yang; Jianping Zhou (45-58).
► A novel superfamily of cysteine-rich peptides (referred to as HsVx1 family) was identified from scorpions. ► The genomic organization of HsVx1 markedly differs from those of other peptides from scorpions. ► The evolution of the HsVx1 superfamily genes were dominated by intron sliding and intron loss. ► Intron in the 3′-terminal region was preferentially lost, whereas that in the 5′-terminal region preferentially slid. ► The reverse complementary sequence of the third intron of an HsVx1-like peptide gene also codes for a different HsVx1-like peptide.
Keywords: Cysteine-rich peptide; Intron gain/loss; Intron sliding; Alternative splicing; Gene recombination; Molecular evolution; Scorpion; Arthropod;

Phylogenetic distribution of protease inhibitors of the Kazal-family within the Arthropoda by Vincent van Hoef; Bert Breugelmans; Jornt Spit; Gert Simonet; Sven Zels; Jozef Vanden Broeck (59-65).
► We performed a data mining study of PSTI-like protein precursors in Arthropoda. ► These were present in the four major arthropod subphyla. ► Sequence analysis revealed a relative conservation. ► This study is evidence for the existence of an ancient protective mechanism.In mammalian pancreatic cells, the pancreatic secretory trypsin inhibitor (PSTI) belonging to the Kazal-family prevents the premature activation of digestive enzymes and thus plays an important role in a protective mechanism against tissue destruction by autophagy. Although a similar protective mechanism exists in Arthropoda, the distribution of these inhibitors in this phylum remains obscure. A comprehensive in silico search of nucleotide databases, revealed the presence of members of the Kazal-family in the four major subphyla of the Arthropoda. Especially in the Hexapoda and the Crustacea these inhibitors are widespread, while in the Chelicerata and Myriapoda only a few Kazal-like protease inhibitors were found. A sequence alignment of inhibitors retrieved in the digestive system of insects revealed a conservation of the PSTI characteristics and strong resemblance to vertebrate PSTI. A phylogenetic analysis of these inhibitors showed that they generally cluster according to their order. The results of this data mining study provide new evidence for the existence of an ancient protective mechanism in metazoan digestive systems. Kazal-like inhibitors, which play an important protective role in the pancreas of vertebrates, also seem to be present in Arthropoda.
Keywords: PSTI; Kazal; Protease inhibitor; Arthropod; Phylogeny;

Octopamine receptor gene expression in three lepidopteran species of insect by Felix Lam; Jeremy N. McNeil; Cam Donly (66-73).
The invertebrate octopaminergic system affects many diverse processes and represents the counterpart to the vertebrate adrenergic/noradrenergic system with the classes of octopamine receptor (OAR) being homologous to those of vertebrate adrenergic receptors. However, there is still little information on the OARs present in different insect species, and the levels and distribution of these receptors throughout the body. cDNAs sharing high similarity with known insect OARs were cloned in three lepidopteran species: the cabbage looper, Trichoplusia ni; the true armyworm, Pseudaletia unipuncta; and the cabbage white, Pieris rapae. Seven major larval tissues and one adult tissue were examined in T. ni using quantitative real-time PCR to determine the relative expression levels of each receptor transcript across different tissues, as well as of all receptor transcripts within individual tissues. A subset of these tissues was also examined in P. unipuncta and P. rapae. All receptor transcripts were expressed in the nervous system of all three species, however, the distribution of the different receptor types varied between species. In all tissues, the OARbeta2 transcript was the most highly expressed, except in the Malpighian tubules where OARbeta1 was highest, and the midgut where there was no significant difference in receptor transcript levels.
Keywords: Octopamine; Receptors; Tyramine; Lepidopteran; Insect; qPCR;

► Ion transport peptide (ITP) elevates intracellular cyclic AMP in the locust ileum. ► Tissue levels of cyclic GMP also increase due to ITP stimulation. ► Factors other than ITP in the corpus cardiacum elevate ileum levels of cyclic AMP. ► Cyclic GMP stimulates fluid reabsorption across the locust ileum in vitro.The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl, K+ and Na+ reabsorption, whereas secretion of H+ into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.
Keywords: Locust; Neuropeptide; Antidiuresis; Ion transport;

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles by Fernando Zúñiga-Navarrete; Isabel Gómez; Guadalupe Peña; Alejandra Bravo; Mario Soberón (81-86).
► Glycosyl–phosphatidyl–inositol (GPI)-anchored proteins are involved in binding of Bacillus thuringiensis Cry3Aa to Tenebrio molitor brush border membranes. ► A T. molitor GPI-anchored alkaline phosphatase ALP binds Cry3Aa toxin. ► The GPI-anchored ALP was preferentially expressed in T. molitor early instar larvae in comparison to late instar larvae. ► GPI-anchored ALP are involved in the mode of action of B. thuringiensis Cry toxins in three different insect orders. Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.
Keywords: Bacillus thuringiensis; Cry toxins; Alkaline phosphatase; GPI-anchored proteins; Toxin–receptor interaction;

► Cyt toxins are pore-forming toxins that affect midgut cells from dipteran insects. ► Cyt toxins synergize the toxicity of some Cry toxins by functioning as receptor for Cry toxins. ► Cyt toxins overcome resistance to Cry toxins in mosquitoes. ► N-terminal region is involved in oligomerization and C-terminal region in membrane insertion. ► Cyt toxin have similarities to related proteins from other bacteria and fungus that are pathogenic to plants or insects, a phylogenetic tree is presented. Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes.
Keywords: Cyt toxin; Bacillus thuringiensis; Synergism;

Anopheles gambiae, Anoga-HrTH hormone, free and bound structure – A nuclear magnetic resonance experiment by Grace Mugumbate; Graham E. Jackson; David van der Spoel; Katalin E. Kövér; László Szilágyi (94-100).
► We have determined the preferred conformation of Anoga-HrTH from Anopheles gambiae, in water and bound to a membrane. ► We report changes in this conformation upon binding to its G-protein coupled receptor. ► The interaction between Anoga-HrTH and its receptor were used to explain structure-activity data. ► The Anoga-HrTH/AKHR complex was stabilized by both polar and hydrophobic interactions involving residues in helices and extracellular loops. ► The estimated free energy of binding (ΔG b ) and inhibition constant (K i ), are −47.3 kJ/mol and 5.2 nM respectively.The spread of malaria by the female mosquito, Anopheles gambiae, is dependent, amongst other things, on its ability to fly. This in turn, is dependent on the adipokinetic hormone, Anoga-HrTH (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH2). No crystal structure of this important neuropeptide is available and hence NMR restrained molecular dynamics was used to investigate its conformational space in aqueous solution and when bound to a membrane surface. The results showed that Anoga-HrTH has an almost cyclic conformation that is stabilized by a hydrogen bond between the C-terminus and Thr3. Upon docking of the agonist to its receptor, this H-bond is broken and the molecule adopts a more extended structure. Preliminary AKHR docking calculations give the free energy of binding to be −47.30 kJ/mol. There is a close correspondence between the structure of the docked ligand and literature structure–activity studies. Information about the 3D structure and binding mode of Anoga-HrTH to its receptor is vital for the design of suitable mimetics which can act as insecticides.
Keywords: Anopheles gambiae; Anoga-HrTH; Molecular dynamics; GROMACS; AUTODOCK;

Evaluation of insect CAP2b analogs with either an (E)-alkene, trans- or a (Z)-alkene, cis-Pro isostere identifies the Pro orientation for antidiuretic activity in the stink bug by Ronald J. Nachman; Xiaodong J. Wang; Felicia A. Etzkorn; Krzysztof Kaczmarek; Janusz Zabrocki; Juan Lopez; Geoffrey M. Coast (101-106).
► CAP2b analogs were synthesized with (Z)-alkene, and (E)-alkene components. ► These components lock in a cis- or trans-Proline orientation, respectively. ► They were tested for antidiuretic activity on stink bug Malpighian tubules. ► Only the trans-Pro analog had antidiuretic activity. ► This is consistent with the Pro in CAP2b adopting a trans orientation.The CAP2b neuropeptide family plays an important role in the regulation of the processes of diuresis and/or antidiuresis in a variety of insects. While Manse-CAP2b (pELYAFPRV-NH2) and native CAP2bs elicit diuretic activity in a number of species of flies, native CAP2b sequences have been shown to elicit antidiuretic activity in the kissing bug Rhodnius prolixus and the green stink bug Acrosternum hilare, the latter being an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either a (Z)-alkene, cis-Pro or an (E)-alkene, trans-Pro isosteric component were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretion by Malpighian tubules isolated from A. hilare. The conformationally constrained trans-Pro analog demonstrated statistically significant antidiuretic activity, whereas the cis-Pro analog failed to elicit activity. The results are consistent with the adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with receptors associated with the antidiuretic process in the stink bug. In addition, the results are further consistent with a theory of ligand-receptor coevolution between the CAP2b and pyrokinin/PBAN neuropeptide classes, both members of the ‘-PRXamide’ superfamily. This work further identifies a scaffold with which to design mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2b-regulated diuretic/antidiuretic functions.
Keywords: Antidiuretic; Stink bug; Peptidomimetic; Neuropeptide; Cyclic;

Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption by Na Yu; Veronica Benzi; Moises João Zotti; Dorien Staljanssens; Krzysztof Kaczmarek; Janusz Zabrocki; Ronald J. Nachman; Guy Smagghe (107-112).
► A series of synthetic insect sulfakinin (SK) analogs was injected in adults of the red flour beetle (Tribolium castaneum) to investigate their actions on food intake. ► Both sulfated and non-sulfated SKs inhibited food intake in T. castaneum adults. ► Substitutions in position 1, 4 and 8 by Ser, Asu and Ala retained the food intake inhibitory activity, while Nle substitution in position 7 led to inactive compounds. ► The Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12) increased food intake, suggesting an action as antagonist.The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.
Keywords: Sulfakinin; Food intake; Tribolium castaneum; Analog; Antagonist;