Peptides (v.26, #1)

Invertebrate neuropeptides V by Harold L. Atwood (1-2).

The primary structures and molecular mass data of neuropeptides that are released from major neuroendocrine sites of the American cockroach, Periplaneta americana, are well known. In the current study we extensively surveyed neuropeptides of the corpora cardiaca, the corpora allata and the abdominal perisympathetic organs, from 14 related cockroach species belonging to the family Blattidae. Mainly, this survey was executed by using mass spectrometric methods (MALDI-TOF MS). Peptides which appeared to be modified, as judged from their mass data and comparison with known data from P. americana, were fragmented by means of the post-source decay technique to determine their amino acid sequence. Single organ preparations sufficed to reveal the peptide pattern in neurohemal organs and to identify species-specific modifications, making it possible to glean information also from tissues of less abundant species that were caught during field trips. The peptide inventory described in this study is very typical of cockroaches of the family Blattidae and clearly separates this taxon from other taxa of cockroaches. The majority of neuropeptides was identical in all investigated species. Some peptides, however, displayed remarkable variations in their sequence which hint to a differential rate of modification among peptides of the same neuropeptide family. These modifications serve to distinguish further relatedness of taxa within the Blattidae.
Keywords: Periplaneta; Blattidae; Insect neuropeptide; Periviscerokinin; FXPRLamide; Mass spectrometry; Evolution;

Neuropeptides associated with the frontal ganglion of larval Lepidoptera by Neil Audsley; June Matthews; Robert J. Weaver (11-21).
The occurrence of neuropeptides in the frontal ganglia of larvae of the tobacco hawkmoth, Manduca sexta, the tomato moth, Lacanobia oleracea and the cotton leafworm, Spodoptera littoralis was investigated using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and enzyme-linked immunosorbent assay (ELISA). Only three types of peptides could be identified or assigned from frontal ganglion extracts; M. sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT), and F/YXFGL-NH2 allatostatins. The peptide profiles of frontal ganglion of L. oleracea and S. littoralis were similar, with ten identical [M + H]+ ions, seven of which could be assigned to known lepidopteran peptides (Manse-AT, cydiastatin 2, 3, 4 and helicostatin 1, 5, 9). In addition, mass ions corresponding to helicostatin 7 (which was confirmed by MALDI-post source decay analysis) and Manse-AS were present in frontal ganglia of L. oleracea and helicostatin 6 in frontal ganglia of S. littoralis. Only four mass ions from M. sexta frontal ganglia corresponded to known peptides, cydiastatin 3 and 4, helicostatin 1, and Manse-AT. The only difference between the profiles of frontal ganglia from different stages of L. oleracea were mass ions which could not be assigned, and no differences were observed in the allatoregulatory peptides present. In HPLC fractions of M. sexta frontal ganglia, F/YXFGL-NH2 allatostatin-like immunoreactivity was widespread suggesting that more allatostatins were present than were identified.
Keywords: Peptidomics; Insect; Lepidoptera; Allatostatin; Allatotropin;

Putative γ-conotoxins in vermivorous cone snails: the case of Conus delessertii by Manuel B. Aguilar; Estuardo López-Vera; Julita S. Imperial; Andrés Falcón; Baldomero M. Olivera; Edgar P. Heimer de la Cotera (23-27).
Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0 Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure (ACKOKNNLCAITγMAγCCSGFCLIYRCS*; O, hydroxyproline; γ, γ-carboxyglutamate; *, amidated C-terminus; calculated average mass, 3169.66 Da) of de7a contains a motif (―γCCS―) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and γ-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin γ-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and γ-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.
Keywords: Conacea; Conidae; Cone snail; Conus delessertii; Gamma-conotoxin; Pacemaker channels;

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus by V.A. Te Brugge; V.C. Lombardi; D.A. Schooley; I. Orchard (29-42).
The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795–804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469–74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795–804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469–74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.
Keywords: Calcitonin-like; DH31; Diuretic hormone; Immunohistochemistry; Neurohemal; Diploptera punctata;

The distribution and activity of tachykinin-related peptides in the blood-feeding bug, Rhodnius prolixus by Rodney Kwok; Doreen Chung; Victoria Te Brugge; Ian Orchard (43-51).
The invertebrate tachykinin-related peptides (TRPs) with the conserved C-terminal sequence FX1GX2Ramide shows sequence similarity to the vertebrate tachykinins after which they are named, and are hypothesized to be ancestrally related. In this study a polyclonal antiserum generated against a locust tachykinin (LomTK I), was used to demonstrate the presence and describe the distribution of LomTK-like immnoreactivity in the CNS and gut of Rhodnius prolixus. Reverse phase high performance liquid chromatography (RP-HPLC) was used in combination with a sensitive radioimmunoassay (RIA) to demonstrate picomolar amounts of immunoreactive material in the CNS, and femptomolar amounts associated with the hindgut. Furthermore, the results from CNS extracts separated by RP-HPLC, suggest that at least two tachykinin isoforms exist in R. prolixus. A hindgut contraction assay was developed to quantify the myotropic effects of selected LomTKs on R. prolixus hindgut contraction. Both LomTK I and II caused an increase in the frequency of hindgut contractions with EC50 values of 3.6 × 10−8  M and 3.8 × 10−8  M for LomTK I and II, respectively.
Keywords: Insect; Immunoreactivity; Radioimmunoassay; Neurons; Neuropeptide; Myotropic;

The presence of APGWamide in Octopus vulgaris: a possible role in the reproductive behavior by Carlo Di Cristo; Jan Van Minnen; Anna Di Cosmo (53-62).
The concerted action of many neuropeptides has been implicated in the nervous control of specific behaviors in many molluscs. In the present study, the presence of amidated tetrapeptide Ala-Pro-Gly-Trp-NH2 (APGWamide) in those lobes that are involved in the control of reproductive behavior in Octopus vulgaris has been investigated. APGWamide immunoreactivity was mainly confined to the posterior olfactory lobule and in the inferior frontal system. These areas are involved in Octopus in the processing of either chemotactile sense or olfaction. From these lobes, immunoreactive fibers reached other lobes of the central nervous system (CNS) which could be indirectly involved in the reproductive behavior. APGWamide immunoreactivity was also present in the glandular cells of the oviducal gland in the female reproductive system. These results constitute the first detailed immunolocalization of APGWamide in cephalopods and open a new insight into the possible effects that both distant and close chemical stimuli can exert on neuropeptidergic circuitries, which may affect the reproductive behavior of cephalopods.
Keywords: Neuropeptide; Olfactory lobe; Oviducal gland; Nervous system; Cephalopods; Chemical stimuli; Olfaction;

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper (Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle (∼35 days), with the maximum response occurring on days 16–18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.
Keywords: Allatotropin; Allatostatin; Corpora allata; Juvenile hormone; Radiochemical assay;

Melatonin-induced neuropeptide release from isolated locust corpora cardiaca by J. Huybrechts; A. De Loof; L. Schoofs (73-80).
A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.
Keywords: Locusta migratoria; Neuropeptides; Corpora cardiaca; Neuropeptide release; Octopamine; Forskolin; Melatonin;

Allatostatins (ASTs) are a family of neuropeptides that inhibit the biosynthesis of juvenile hormone in cockroaches and related insects, but not in flies. Two receptors for allatostatins, DAR-1 and DAR-2, with sequence similarity to mammalian galanin receptors have previously been cloned in Drosophila melanogaster. To study the distribution of the predicted DAR-2 protein by immunocytochemistry, antisera were raised against a synthetic peptide corresponding to part of the amino terminus of the receptor sequence. In the brain of larval Drosophila, immunoreactivity appeared to be associated with glial septa surrounding neuropil compartments. In the ventral ganglion, immunoreactive cell bodies appeared to reside in the cortex of the ganglion, surrounding the central neuropil and neurohemal organs. In addition, double labeling immunocytochemistry revealed a substantial superposition between distribution of AST-like immunoreactivity and the putative DAR-2 protein in at least five cell bodies in the region of the ring gland corresponding to the corpora cardiaca.
Keywords: Drosophila melanogaster; Allatostatin; Allatostatin receptor; Co-localization;

A study of signal transduction for the two diuretic peptides of Diploptera punctata by S.S. Tobe; J.R. Zhang; D.A. Schooley; G.M. Coast (89-98).
We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.
Keywords: Diuretic hormones; Diploptera punctata; Signal transduction; Camp; cGMP;

The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50  ∼ 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF > Aea-NPF > Drm-NPF > Drm-NPF8–36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito.
Keywords: Insect; Diptera; Anopheles; Malaria; G protein-coupled receptor; Neuropeptide F; Neuropeptide Y;

Pharmacology of stomoxytachykinin receptor depends on second messenger system by Jeroen Poels; Ronald J. Nachman; Karl E. Åkerman; Hendrica B. Oonk; Felix Guerrero; Arnold De Loof; Anna E. Janecka; Herbert Torfs; Jozef Vanden Broeck (109-114).
STKR is a neurokinin receptor derived from the stable fly, Stomoxys calcitrans. Insect tachykinin-related peptides, also referred to as “insectatachykinins”, produce dose-dependent calcium and cyclic AMP responses in cultured Drosophila melanogaster Schneider 2 (S2) cells that were stably transfected with the cloned STKR cDNA. Pronounced differences in pharmacology were observed between agonist-induced calcium and cyclic AMP responses. The results indicate that the pharmacological properties of STKR depend on its coupling to a unique second messenger system. Therefore, a model postulating the existence of multiple active receptor conformations is proposed. This article presents the first evidence that an insect peptide receptor with dual coupling properties to second messenger systems can display agonist-dependent functional differences.
Keywords: Tachykinin; G protein-coupled receptor; Stable fly; Schneider 2 cells; Pharmacology; Cyclic AMP; Ca2+;

Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects by Ronald J. Nachman; Tom Vercammen; Howard Williams; Krzysztof Kaczmarek; Janusz Zabrocki; Liliane Schoofs (115-120).
The aliphatic amino diacid α-aminosuberic acid can function as an effective, stable mimic of the hydrolysis-susceptible Tyr(SO3H) group in sulfakinin neuropeptide analogs for both hindgut contractile activity in cockroach and food intake-inhibition activity in the desert locust. In the analog, the acidic sulfate group is replaced with an acidic carboxyl group. The degree of activity of sulfakinin analogs is correlated with the carboxyl/α-carbon distance in the cockroach hindgut contractile assay. The results represent an important step in the design and synthesis of biostable, sulfakinin analogs that could potentially suppress the feeding behavior of destructive insect pests of agricultural importance.
Keywords: Insect neuropeptide; Cockroach; Locust; Pest management; Myotropic;

Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness by Scott F. Cummins; Catherine H. Schein; Yuan Xu; Werner Braun; Gregg T. Nagle (121-129).
The marine mollusk Aplysia releases the water-borne pheromone attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica attractin contains two antiparallel α-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of attractin abolishes attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.
Keywords: Albumen gland; Aplysia; Attractin; Bursatella leachii; Mollusk; Protein pheromone;

Were arachnids the first to use combinatorial peptide libraries? by Brianna L. Sollod; David Wilson; Olga Zhaxybayeva; J. Peter Gogarten; Roger Drinkwater; Glenn F. King (131-139).
Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.
Keywords: Peptide toxin; Toxin evolution; Combinatorial peptide library; Spider; Scorpion; Arachnid; Cone snail; Atracotoxin; Conotoxin; Prepropeptide; Toxin precursor; Cysteine scaffold;

Contents Volume 25 (153-167).