Ecdysiostatins and allatostatins in Schistocerca gregaria.
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Amino Acid Sequence↗
Biomedical subjects
L Schoofs
Publications and source records attributed to L Schoofs.
Immunocytochemical distribution of angiotensin I-converting enzyme-like immunoreactivity in the brain and testis of insects.
Angiotensin converting enzyme (ACE) is Zn2+ metallopeptidase which plays an important role in blood pressure homeostasis in mammals and other vertebrates. Homologues of ACE involved in the biosynthesis of mammalian peptide hormones have also been identified in the insects, Musca domestica, Drosophila melanogaster and Haematobia irritans exigua. In the pursuit of the biological role of insect ACE, this work focused on the tissue and cellular distribution of ACE in several insect species. The localisation of ACE in the central nervous system and reproductive tissues from a number of insect species suggests that ACE is of physiological importance in these tissues. By means of an antiserum to housefly ACE, we found that ACE-like immunoreactivity was abundantly present in the neuropil areas of the brain of all insects investigated, suggesting a role for ACE in the metabolic inactivation of peptide neurotransmitters. Especially in the fleshfly, Neobellieria bullata neuropile staining is abundant. In the cockroach Leucophaea maderae, immunoreactive staining was abundant in the neuronal perikarya as well as in the neuropilar regions. Staining in neurosecretory cells was also observed in the brains of the lepidopteran species, Bombyx mori and Mamestra brassica. The localisation of ACE in neurosecretory cells is consistent with the role as a processing hormone, involved in the generation of active peptide hormones. ACE was found to be co-localised with peptides of the FXPRLamide family in M. brassica and in B. mori, suggesting a role for the biosynthesis of these hormones. Finally, we found ACE-like immunoreactivity in the testis of Locusta migratoria, N. bullata and Leptinotarsa decemlineata, providing additional evidence for its important role in insect reproduction.
Animals↗
A novel peptide-processing activity of insect peptidyl-dipeptidase A (angiotensin I-converting enzyme): the hydrolysis of lysyl-arginine and arginyl-arginine from the C-terminus of an insect prohormone peptide.
Insect peptidyl-dipeptidase A [angiotensin I-converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian germinal ACE. Mammalian somatic ACE is important in blood homoeostasis, but the role of ACE in insects is not known. Immunocytochemistry has been used to localize ACE in the neuroendocrine system of the locust, Locusta migratoria. Staining was observed in five groups of neurosecretory cells in the brain and suboesophageal ganglion, in the nervi corpori cardiaci, the storage part of the corpora cardiaca and in the nervi corpori allati. In three groups of neurosecretory cells, ACE co-localized with locustamyotropins, suggesting a possible role for the enzyme in the metabolism of these neuropeptides. We demonstrate in vitro a novel activity of ACE that removes pairs of basic amino acid residues from a locustamyotropin peptide extended at the C-terminus with either Gly-Lys-Arg or Gly-Arg-Arg, corresponding to a consensus recognition sequence for endoproteolysis of prohormone proteins by prohormone convertases. The low Km and high kcat values (Km 7.3 and 5.0 microM, kcat 226 and 207 s-1 for the hydrolysis of Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg, respectively) obtained for the hydrolysis of these two peptides by insect ACE means that these peptides, along with mammalian bradykinin, are the most favoured in vitro ACE substrates so far identified. The discovery of this in vitro prohormone-processing activity of insect ACE provides a possible explanation for the intracellular co-localization of the enzyme with locustamyotropin peptides, and provides evidence for a new role for ACE in the biosynthesis of peptide hormones and transmitters.
Animals↗
The ovary of the desert locust Schistocerca gregaria contains a glycine- and proline-rich peptide that displays sequence similarities with a new class of GPRP proteins from plants.
A novel, highly hydrophobic, glycine- and proline-rich peptide was characterized in the ovary of the desert locust, Schistocerca gregaria. The peptide was detected as one of the major peaks in a chromatographic separation of an acidic methanolic extract of 50 mature ovaries. Electrospray mass spectrometry yielded a molecular mass of 6305 Da. The partial amino acid sequence as determined by Edman degradation based automated microsequencing is as follows: Ala-Tyr-Pro-Ala-His-Gln-Gly-Tyr- Pro-Ala-His-Val-Gly-Tyr-Ala-Arg-Val-Gly-Tyr-Gly- Gly-Tyr-Pro-Ser-Tyr-Gly-Tyr-Pro-Ala. Four amino acids (Gly, Pro, Ala, and Tyr) account for more than 80% of the composition of this sequence. Gly-Tyr-Pro is the most important repetitive motif. Ala-Tyr-Pro, Gly-Tyr-Gly and Gly-Tyr-Ala occur as variations of this motif. The novel glycine- and proline-rich insect peptide displays structural characteristics similar to those of a new class of glycine- and proline-rich proteins (GPRP) that have recently been identified in Arabidopsis thaliana (thale cress) and Daucus carota (carrot). The GPRP of A. thaliana contains the same repetitive motifs (except for Ala-Tyr-Pro), the Gly-Tyr-Pro motif also being the most abundant.
Amino Acid Sequence↗
Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria.
The ovary of the desert locust, Schistocerca gregaria, contains multiple inhibitors of serine proteases. Five serine protease inhibitors, designated SGPI-1-5 (Schistocerca gregaria protease inhibitors) were purified from methanolic extracts of mature ovaries and analyzed by mass spectrometry and amino acid sequencing. The revealed primary structures display amino acid similarities and are related to the serine protease inhibitors identified in the hemolymph of Locusta migratoria. All inhibitors show an in vitro inhibiting activity towards alpha-chymotrypsin. In addition, SGPI-1 displays in vitro inhibiting activity towards trypsin, and SGPI-2 is a potent pancreatic elastase inhibitor. Differences in inhibitory specificities towards the locust endogenous serine proteases can be readily attributed to the amino acid sequence within the active region and also to amino acid residues beyond the P1-P'1 bond. A difference in one or two amino acid residues around the reactive sites results in considerable alteration of the inhibitory specificity. The temporal and spatial distribution of SGPI-1-5 was studied by RP-HPLC analysis. All inhibitors occur in hemolymph, ovaries, testes and fat body of adults but are absent in the gut. They are also present in larval hemolymph and fat body. An antibody raised against SGPI-2 shows positive immunostaining in the ovarian follicle cells.
Amino Acid Sequence↗
Angiotensin II and angiotensin-converting enzyme as candidate compounds modulating the effects of testis ecdysiotropin in testes of the gypsy moth, Lymantria dispar1.
Lymantria dispar testes synthesize immunodetectable ecdysteroid in vitro in response to the brain peptide, testis ecdysiotropin (TE), acting primarily via a cascade involving Gi protein, diacyl glycerol, and phosphokinase C. However, a component of TE activation also involves the opposite cascade, Gs protein, cAMP, and phosphokinase A. Excess cAMP inhibits the action of TE, acting as a feedback modulator. Here, we show that bovine angiotensin II (AII) and bovine angiotensin converting enzyme (ACE) act like cAMP, inducing synthesis of immunodetectable ecdysteroid by pupal testes in vitro, but are antagonistic to coincubated TE. In addition, an insect ACE antibody clearly stains the spermatogenic cells through all stages of development, as well as testis sheath tissue where ecdysteroid is synthesized. AII induces synthesis of cAMP by pupal testes in vitro. Therefore, insect homologs of mammalian AII and ACE are good candidates for the peptides responsible for the cAMP cascade and as modulators of TE action in lepidopteran testes. Saralasin, an analog of AII that blocks angiotensin receptors in mammals, behaved like AII in inducing ecdysteroid secretion with ecdysteroidogenic effects additive to either angiotensin or ACE. Therefore, the receptors for the insect form of angiotensin on lepidopteran testis cells are probably different from those in mammals. Saralasin also inhibited ecdysteroid synthesis when combined with TE, as did AII.
Angiotensin II↗
Insect larvae contain substances toxic to adults: the discovery of paralysins.
Acidic methanolic extracts of larvae of nine different insect species were found to contain substances that cause a lethal effect in the adult stage of the same species and of other species. These endogenous toxic substances, apparently being widely spread over the class of insects, were designated as paralysins, because of their immediate and observable paralytic effect upon injection. The developmental concentration curves of five different species of insects (Galleria mellonella (Lepidoptera), Neobellieria bullata (Diptera), Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Schistocerca gregaria (Orthoptera) indicate that the toxins are not present throughout all the developmental stages in the same concentration. The strongest paralytic activity was found in late instar larvae or in the early pupal stage. The temporal distribution of paralysins during development suggests that they might be involved in metamorphosis.
Journal Article↗
Peptidergic control of the corpus cardiacum-corpora allata complex of locusts.
The brain-corpora cardiaca-corpora allata complex of insects is the physiological equivalent of the brain-hypophysis axis of vertebrates. In locusts there is only one corpus cardiacum as a result of fusion, while most other insect species have a pair of such glands. Like the pituitary of vertebrates, the corpus cardiacum consists of a glandular lobe and a neurohemal lobe. The glandular lobe synthesizes and releases adipokinetic hormones. In the neurohemal part many peptide hormones, which are produced in neurosecretory cells in the brain, are released into the hemolymph. The corpora allata, which have no counterpart in vertebrates, synthesize and release juvenile hormones. The control of the locust corpus cardiacum-corpora allata complex appears to be very complex. Numerous brain factors have been reported to have an effect on biosynthesis and release of juvenile hormone or adipokinetic hormone. Many neuropeptides are present in nerves projecting from the brain into the corpora cardiaca-corpora allata complex, the most important ones being neuroparsins, ovary maturating parsin, insulin-related peptide, diuretic peptide, tachykinins, FLRFamides, FXPRLamides, accessory gland myotropin I, crustacean cardioactive peptide, and schistostatins. In this paper, the cellular distribution, posttranslational processing, peptide-receptor interaction, and inactivation of these peptides are reviewed. In addition, the signal transduction pathways in the release of adipokinetic hormone and juvenile hormone from, respectively, the corpora cardiaca and corpora allata are discussed.
Animals↗
Biological activity of structural analogs and effect of oil as a carrier of trypsin modulating oostatic factor of the gray fleshfly Neobellieria bullata.
The trypsin modulating oostatic factor from the gray fleshfly Neobellieria bullata (Neb-TMOF) is released from the ovary at the end of vitellogenesis and inhibits trypsin biosynthesis in the midgut. This inhibition indirectly results in an arrest of oocyte growth. Additional experiments with N. bullata were performed to characterize its trypsin modulating and oostatic properties in more detail. After suspending the peptide in wheat germ oil, the threshold dose for oostatic activity was lowered one thousand times (2.10(-5) in oil versus 2.10(-2) pmoles per fly in Ringer). By use of the Neobellieria trypsin biosynthesis assay, 17 analogs of the hexapeptide were tested for inhibitory activity. The following structural elements were demonstrated to be critical for biological activity: the alcohol function at position 3 (Thr residue); a positively charged basic group at the C terminus (His residue); and the Asn side chain at positions 1 and 4.
Amino Acid Sequence↗
Isolation and immunocytochemical characterization of three tachykinin-related peptides from the mosquito, Culex salinarius.
Three myotropic peptides belonging to the Arg-amide insect tachykinin family were isolated from whole-body extracts of the mosquito, Culex salinarius. The peptides, APSGFMGMR-NH2, APYGFTGMR-NH2 and APSGFFGMR-NH2 (designated culetachykinin I, II, and III) were isolated and purified on the basis of their ability to stimulate muscle contractions of isolated Leucophaea maderae hindgut. Biologically inactive methionine sulfoxides of two of the three peptides were isolated using an ELISA system based upon antiserum raised against APYGFTGMR-NH2 and identified with mass spectrometry. Immunocytochemistry localized these peptides in cells in the brain, antennae, subesophageal, thoracic and abdominal ganglion, proventriculus and midgut. Nerve tracts containing these peptides were found in the median nerve of the brain, central body, nervi corpus cardiaci, cervical nerve, antennal lobe and on the surface of the midgut.
Amino Acid Sequence↗
Identification of two novel peptides from the central nervous system of the desert locust, Schistocerca gregaria.
From an acid methanolic extract of about 7000 brains of the desert locust (Schistocerca gregaria) two novel neuropeptides, schistomyotropin-1 (Scg-MT-1) and schistomyotropin-2 (Scg-MT-2), were isolated and identified. Their primary structures are GAAPAAQFSPRLamide (Scg-MT-1) and TSSLFPHPRLamide (Scg-MT-2). Scg-MT-1 belongs to the locustamyotropin family characterized by its FXPRLamide C-terminus. Scg-MT-2 has a similar carboxyl end with the F-residue one position further away from the C-terminus. This may account for its being 10 times less active then Scg-MT-1 in stimulating cockroach hindgut motility.
Animals↗
Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria.
A protease-inhibitor was isolated from mature ovaries of Schistocerca gregaria by a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development.
Animals↗
False positive immunostaining of Locusta neurosecretory cells with a variety of preimmune sera.
A large number of antisera directed against vertebrate neuropeptides have been reported to yield positive staining when applied to insect brains. In most cases, the preimmune serum of the same animal in which the antiserum was developed is not available for testing in control experiments. We have experienced that a large percentage of preimmune sera, as well as a culture medium for hybridomas, stain cell populations and fibers in the central nervous system of the insect Locusta migratoria. Purification of these preimmune sera on a Protein A and Protein G support indicates that the reaction is due to preexisting antibodies of the IgG class. Western analysis of brain and nervous tissue extracts indicates the presence of two immunoreactive 27-kDa bands. These bands could also be visualized in other tissue extracts such as muscle, midgut, Malpighian tubules, and fat body of Locusta. The brain of other insect species, such as Periplaneta americana, Leucophaea maderae, and Neobellieria bullata were devoid of the false immunopositive reaction. There is no easy way to eliminate this type of immunoreaction. It follows that when affinity chromatographic purification of the antibody is not feasible, it is essential to include in the control procedure, the preimmune serum of the animal that was used for the production of the antiserum. This means that it should become common practice to sell or exchange sera together with their corresponding preimmune sera.
Animals↗
Peptides in the locusts, Locusta migratoria and Schistocerca gregaria.
The first peptide identified in locusts was adipokinetic hormone I (AKH-I), a neurohormone mobilizing lipids from the fat body. No other locusts peptides were isolated until 1985. From then on peptide identification started to boom at such a tremendously fast rate that even specialists in the field could hardly keep track. At this moment the total number of different insect neuropeptide sequences exceeds 100. Currently, the locusts Locusta migratoria and Schistocerca gregaria are the species from which the largest number of neuropeptides has been isolated and sequenced, namely 56. Myotropic bioassays have played a major role in the isolation and subsequent structural characterization of locust neuropeptides. They have been responsible for the discovery of locustamyotropins, locustapyrokinins, locustatachykinins, locustakinin, locusta accessory gland myotropins, locustasulfakinin, cardioactive peptide, and locustamyoinhibiting peptides. Members of the myotropin peptide families have been associated with a variety of physiological activities such as myotropic activities, pheromonotropic activities, diapause induction, stimulation of cuticular melanization, diuresis, pupariation, and allatostatic activities. Recently, we have identified in Schistocerca 10 peptides belonging to the allatostatin peptide family, which inhibit peristaltic movements of the oviduct. Some of the myotropins appear to be important neurotransmitters or modulators innervating the locust oviduct, the salivary glands, the male accessory glands, and the heart, whereas others are stored in neurohemal organs until release in the hemolymph. Some myotropic peptides have been found to be releasing factors of neurohormones from the corpora cardiaca. Several peptides isolated in locusts appear to be unique to insects or arthropods; others seem to be members of peptides families spanning across phyla: two vasopressin-like peptides, FMRFamide-related peptides, Locusta diuretic hormone (CRF-like), Locusta insulin-related peptide, locustatachykinins, locustasulfakinin (gastrin/CCK-like). In a systematic structural study of neuropeptides in Locusta, several novel peptides have been isolated from the corpora cardiaca and the pars intercerebralis. They include the neuroparsins, two 6-kDa dimeric peptides, and three proteinase inhibitors. Ovary maturating parsin is the first gonadotropin identified in insects. The isolation of a peptide from an ovary extract that inhibits ovary maturation in Schistocerca gregaria is currently underway in our lab. The proteinase inhibitors, recently found to be mainly transcribed in the fat body, are believed to play a role in defense reactions of insects. Finally, a locust ion transport peptide and a peptide stimulating salivation recently can be added to this extensive list of locust peptides.
Amino Acid Sequence↗
Isolation and characterization of an adipokinetic hormone release-inducing factor in locusts: the crustacean cardioactive peptide.
A methanolic extract of 7000 desert locust (Schistocerca gregaria) brains contains several factors that stimulate the in vitro release of adipokinetic hormone (AKH) by glandular cells of locust (Locusta migratoria and Schistocerca gregaria) corpora cardiaca. The most potent one has now been fully identified. Matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis revealed a mass of 954.6 Da. The primary structure of the peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, appeared identical to that of a previously identified crustacean cardioactive peptide. This myotropin was first isolated from the shore crab, Carcinus maenas, and later from several insect species, but was never reported in the context of AKH release. The present study shows that synthetic crustacean cardioactive peptide induces the release of AKH from corpora cardiaca in a dose-dependent manner when tested in concentrations ranging from 10(-5)-10(-9) M. This is the first demonstration in invertebrates of a peptide neurohormone controlling the release of a second peptide hormone.
Animals↗
Isolation and characterization of schistostatin-2(11-18) from the desert locust, Schistocerca gregaria: a truncated analog of schistostatin-2.
An octapeptide was isolated from 7000 brains of the desert locust. Schistocerca gregaria by screening of HPLC fractions using a RIA for Dip-AST-2 (allatostatin-2 from the cockroach). Maldi-TOF-MS revealed a mass of 921.4 Da. The primary structure of the peptide is LPVYNFGL-NH2. It is identical to the C-terminal portion of schistostatin-2 from Schistocerca gregaria. Therefore, it was designated Scg-AST-2(11-18). The chromatographic properties of the synthetic peptide are identical to these of the native peptide. The peptide is a truncated product of Scg-AST-2, suggesting that an endopeptidase which cleaves between Arg and Leu is present in the brain complex of S. gregaria. Although, Scg-AST-2(11-18) contains the same C-terminus as Dip-AST-2, it has no inhibitory activity on the corpora allata (CA) of 2-day-old virgin females of D. punctata. This suggests that Scg-AST2 (11-18) may be the result of a proteolytic inactivation mechanism and/or that it may be involved in stage-dependent down regulation of allatostatic activity. To our knowledge, Scg-AST-2 is the first isolated peptide which has the active core of the allatostatin peptide family but nevertheless shows no activity in this bioassay.
Amino Acid Sequence↗
Isolation and characterization of eight myoinhibiting peptides from the desert locust, Schistocerca gregaria: new members of the cockroach allatostatin family.
Eight myoinhibiting peptides were purified by high performance liquid chromatography from a methanolic extract of 7000 brains of the desert locust, Schistocerca gregaria. Complete sequences were obtained via a novel, combined approach employing: (1) chemical microsequencing and (2) post-source decay analysis on a reflectron time-of-flight mass spectrometer using matrix-assisted laser desorption/ionisation. Each of the peptides shows C-terminal amino acid sequence similarity to cockroach and cricket allatostatins and to blowfly callatostatins. Therefore, these novel peptides were designated Schistocerca gregaria allatostatins (Scg-ASTs) or schistostatins and their primary structures were determined to be: Ala-Tyr-Thr-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (Scg-AST-2), Ala-Thr-Gly-Ala-Ala-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-3), Gly-Pro-Arg-Thr-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-4), Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-5), Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-6), Ala-Gly-Pro-Ala-Pro-Ser-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-7), Glu-Gly-Arg-Met-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-8), and Ala-Pro-Ala-Glu-His-Arg-Phe-Ser-Phe-Gly-Leu-NH2 (Scg-AST-10). Synthetic Scg-AST peptides inhibit the peristaltic movements of the oviduct of S. gregaria. Although all eight peptides show potent inhibitory effects on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diploptera punctata, no allatostatic effects were observed on CA of the desert locust (S. gregaria).
Amino Acid Sequence↗
Immunocytochemical distribution of locustamyoinhibiting peptide (Lom-MIP) in the nervous system of Locusta migratoria.
Locustamyoinhibiting peptide (Lom-MIP) is one of the 4 identified myoinhibiting neuropeptides, isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion complexes of the locust, Locusta migratoria. An antiserum was raised against Lom-MIP for use in immunohistochemistry. Locustamyoinhibiting peptide-like immunoreactivity (Lom-MIP-LI) was visualized in the nervous system and peripheral organs of Locusta migratoria by means of the peroxidase-antiperoxidase method. A total of 12 specific immunoreactive neurons was found in the brain. Processes of these neurons innervate the protocerebral bridge the central body complex and distinct neuropil areas in the proto- and tritocerebrum but not in the deuterocerebrum nor in the optic lobes. The glandular cells of the corpora cardiaca, known to produce adipokinetic hormones, are contacted by Lom-MIP-LI fibers. The corpora allata were innervated by the nervus corporis allati I containing immunoreactive fibers. Lom-MIP-LI cell bodies were also found in the subesophageal ganglion, the metathoracic ganglion and the abdominal ganglia I-IV. In peripheral muscles, Lom-MIP-LI fibers innervate the heart, the oviduct, and the hindgut. In the salivary glands, Lom-MIP-LI was detected in the intracellular ductule of the parietal cells. Possible functions of Lom-MIP are discussed.
Animals↗