Chemical Properties and Physiological Actions of Crustacean Chromatophorotropins

The crustacean pigment-translocating hormones, the red pigment-concentratinghormone (RPCH), an octapeptide, and the light-adapting distalretinal pigment hormone (DRPH), an octadecapeptide, are thefirst invertebrate neurohormones to be fully characterized.Studies with both purified and synthetic hormones show that,in certain decapods, RPCH is a general pigment-concentratinghormone (PCH), affecting the pigments of all kinds of chromatophores(erythrophores, xanthophores, leucophores and melanophores);the DRPH seems to serve not only light-adapting function, butalso act as a general chromatophore pigment-dispersing hormone(PDH). The two hormones thus function as antagonists when regulatingthe color-adaptation of the decapod crustaceans. PCH activityis widely distributed within the arthropod endocrine systems.The first characterized insect neurohormones, the locust adipokinetichormones (AKH I and AKH II), show close structural similaritiesto the crustacean hormone, indicating a common evolution ofsome of the arthropod neurohormones. Physiological studies ofthe three hormones (RPCH, AKH I, and AKH II) and their syntheticanalogs show that they crossreact, i.e., they all exhibit pigment-concentratingactivity when tested on decapod crustaceans, adipokinetic activitywhen tested on locusts, and hyperglycemic activity when testedon cockroaches, although each of the hormones is more potentin its own system. Structure-function studies show, however,that quite different binding-site requirements exist for thehormones in activating their receptors on the various targettissues. The physiological specificity in their action thereforeseems to depend on a differential evolution of the hormone receptors.     

Sequence analyses of adipokinetic hormones II from corpora cardiaca of Schistocerca nitans, Schistocerca gregaria, and Locusta migratoria by fast atom bombardment mass spectrometry

Structures of the second adipokinetic hormones (AKH II's) from three locust species have been assigned by fast atom bombardment mass spectrometry. The AKH II hormone is identical in two Schistocerca species, S. nitans and S. gregaria, but is different in Locusta migratoria. Both AKH II's are related to red pigment-concentrating hormone (RPCH) from prawns, Schistocerca AKH II being [Thr6]-RPCH and Locusta AKH II being [Ala6]-RPCH. Schistocerca AKH II is also bioactive in Locusta individuals.     

The potential of adipokinetic hormone to teach us about neuropeptides

Genetic manipulation and purification techniques are facilitating research into the biology of arthropod neuropeptides. The red pigment concentrating hormone (RPCH)/ adipokinetic hormone (AKH) family are a conserved group of peptides which were first recognized for their hormonal activities. Biosynthesis of AKH in the grasshopper seems to proceed via a large protein precursor (12 kDa) in the cell body of the neuron, which implies precise coordination of synthesis and neural activity. Beginning with a 10 amino acid stretch of known precursor protein sequence, an oligonucleotide primer approach is being used to clone a recombinant AKH gene. Once achieved, this will allow fundamental questions on the biochemistry and physiology of neuropeptides to be addressed. Such work in an arthropod system raises the intriguing possibility of studying neuropeptide level and synthetic activity in single, defined neurons.     

The adipokinetic hormone family in Chrysomeloidea: structural and functional considerations

The presented work is a hybrid of an overview and an original research paper on peptides belonging to the adipokinetic hormone (AKH) family that are present in the corpora cardiaca of Chrysomeloidea. First, we introduce the AKH/red pigment-concentrating hormone (RPCH) peptide family. Second, we collate the available primary sequence data on AKH peptides in Cerambycidae and Chrysomelidae, and we present new sequencing data (from previously unstudied species) obtained by liquid-chromatography coupled with ion trap electrospray ionisation mass spectrometry. Our expanded data set encompasses the primary structure of AKHs from seven species of Cerambycidae and three species of Chrysomelidae. All of these species synthesise the octapeptide code-named Peram-CAH-I (pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp amide). Whereas this is the sole AKH peptide in Cerambycidae, Chrysomelidae demonstrate a probable event of AKH gene duplication, thereby giving rise to an additional AKH. This second AKH peptide may be either Emppe-AKH (pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp amide) or Peram-CAH-II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp amide). The peptide distribution and structural data suggest that both families are closely related and that Peram-CAH-I is the ancestral peptide. We hypothesise on the molecular evolution of Emppe-AKH and Peram-CAH-II from the ancestral peptide due to nonsynonymous missense single nucleotide polymorphism in the nucleotide coding sequence of prepro-AKH. Finally, we review the biological significance of the AKH peptides as hyperprolinaemic hormones in Chrysomeloidea, i.e. they cause an increase in the circulating concentration of proline. The mobilisation of proline has been demonstrated during flight in both cerambycid and chrysomelid beetles.     

Structure-function studies on red pigment-concentrating hormone, II. The significance of the C-terminal tryptophan amide

The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH.     

A novel adipokinetic peptide in a water boatman (Heteroptera, Corixidae) and its bioanalogue in a saucer bug (Heteroptera, Naucoridae)

The corpora cardiaca (CC) of two water bug species, the water boatman Corixa punctata and the saucer bug Ilyocoris cimicoides, contain a substance that cause hyperlipemia in the migratory locust. The primary sequence of one octapeptide belonging to the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family was deduced from the multiple MS(N) electrospray mass data of CC material from each species. Whereas the saucer bug contains the known octapeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Anaim-AKH, the water boatman has a novel peptide identified as pGlu-Leu/Ile-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Corpu-AKH. The ambiguity about the amino acid at position 2, i.e. Leu or Ile, in Corpu-AKH was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with the synthetic peptide containing Leu at position 2. Functionally, the peptides regulate lipid mobilization, as evidenced by an adipokinetic effect after injecting synthetic Anaim-AKH and Corpu-AKH into the respective acceptor species. Swimming activity of I. cimicoides also causes hyperlipemia.     

Physiological actions by hypertrehalosemic hormone and adipokinetic peptides in adult Blaberus discoidalis cockroaches

Members of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family characteristically cause metabolite mobilization by the insect fat body. The present study identified several additional physiological actions in adult Blaberus discoidalis cockroaches that were influenced by synthetic Blaberus hypertrehalosemic hormone (HTH) and other AKH/RPCH family peptides. HTH elevated blood carbohydrate by 4-fold and cytochrome heme a + b synthesis of fat body mitochondria by 3-fold. Both carbohydrate and heme synthesis were dose-responsive to HTH. Carbohydrate synthesis was 10 times more sensitive to HTH than heme synthesis. Heme synthesis was also stimulated by Periplaneta cardioacceleratory hormones (CAH)-I and -II and RPCH but not by AKH-I or -II, at the doses tested. HTH showed strong cardioexcitatory activity. Long-term treatment of decapitated female B. discoidalis with juvenile hormone analog (JHA = methoprene) stimulated by 2.6-fold the rate of synthesis of secreted fat body proteins. HTH enhanced the JHA-dependent export protein synthesis by 42% above that observed with JHA alone. SDS-PAGE demonstrated that JHA determined the nature of the newly synthesized polypeptides; HTH enhanced their synthesis rate. Neither AKH-I nor HTH affected protein synthesis when added directly to isolated fat body. These results demonstrate that peptides of the AKH/RPCH family have multiple physiological actions related to fat body energy metabolism.     

The first decapeptide adipokinetic hormone (AKH) in Heteroptera: a novel AKH from a South African saucer bug, Laccocoris spurcus (Naucoridae, Laccocorinae)

A novel peptide of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family has been elucidated by mass spectrometry from the corpora cardiaca of an African saucer bug species, Laccocoris spurcus. It is the first decapeptide member found in the species-rich taxon Heteroptera, has the primary sequence pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Gly amide and is denoted as Lacsp-AKH. The first eight amino acids are identical to the octapeptide Anaim-AKH of the European saucer bug, Ilyocoris cimicoides. The synthetic peptide Lacsp-AKH elevates lipids upon injection into the hemolymph of L. spurcus at a low dose of 3 pmol. Swimming activity in this saucer bug also causes a significant increase in the lipid concentration in the hemolymph. Thus, both results point to an apparent function of the endogenous new decapeptide Lacsp-AKH in L. spurcus, namely, to regulate lipid mobilization. Isolation of an AKH peptide from the corpora cardiaca of the water bug Aphelocheirus aestivalis (Aphelocheiridae) resulted in the assignment of the octapeptide Anaim-AKH, supporting current phylogenies on the infraorder Nepomorpha.     

Preparation of an 125I-labeled red pigment-concentrating hormone analog.

The red pigment-concentrating hormone (RPCH) from crustacea is an octapeptide with the following structure: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH₂, [4-Tyr]RPCH has been synthesized and was 4 times as active as RPCH in the Leander assay. Iodination of the hormone analog using chloramin-T, lactoperoxidase, solid-state lactoperoxidase, and thallium chloride was carried out and the results were compared. ¹²⁵I-labeled [4-Tyr]RPCH was prepared using a modificated chloramin-T method and was purified from unlabeled and diiodinated peptide by chromatography on Sephadex LH-20.     

The adipokinetic hormones of Heteroptera: a comparative study

The adipokinetic hormones (AKHs) from 15 species of heteropteran Hemiptera (encompassing eight families, six superfamilies and three infraorders) have been isolated and structurally identified using liquid chromatography coupled with mass spectrometry. None of the structures are novel and all are octapeptides. These peptide sequence data are used, together with the previously available AKH sequence data on Heteroptera, to create a larger dataset for comparative analyses. This results, in total, in AKH sequences from 30 species (spanning 13 families), which are used in a matrix confronted with the current hypotheses on the phylogeny of Heteroptera. The expanded dataset shows that all heteropterans have octapeptide AKHs; three species have two AKHs, whereas the overwhelming majority have only one AKH. From a total of 11 different AKH peptides known from Heteroptera to date, three AKHs occur frequently: Panbo‐red pigment‐concentrating hormone (RPCH) (×10), Schgr‐AKH‐II (×6) and Anaim‐AKH (×4). The heteropteran database also suggests that particular AKH variants are family‐specific. The AKHs of Heteroptera: Pentatomomorpha (all terrestrial) are not present in Nepomorpha (aquatic) and Gerromorpha: Gerridae (semiaquatic); AKHs with a Val in position 2 are absent in the Pentatomomorpha (only AKHs with Leu² are present), whereas Val² predominates in the nonterrestrial species. An unexpected diversity of AKH sequences is found in Nepomorpha, Nepoidea, Nepidae and Nepinae, whereas Panbo‐RPCH (which has been identified in all infraorders of decapod crustaceans) is present in all analysed species of Pentatomidae and also in the only species of Tessaratomidae investigated. The molecular evolution of Heteroptera with respect to other insect groups and to crustaceans is discussed     

Vanessa cardui adipokinetic hormone (Vanca-AKH) in butterflies and a moth

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.     

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.     

Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

Crustacean Pigmentary-Effector Hormones: Chemistry and Functions of RPCH, PDH, and Related Peptides

Integumental color changes and eye pigment movements in crustaceansare regulated by pigmentary-effector hormones. The identifiedhormones include: an octapeptide RPCH (red pigment-concentratinghormone) and several forms of octadecapeptide PDH (pigment-dispersinghormone: -PDH, ß-PDH). RPCH-related peptides (AKHs,adipokinetic hormones) and PDH-related peptides (PDFs, pigment-dispersingfactors) occur in insects, and are recognized as members ofAKH/RPCH and PDH/PDF peptide families. The domain for maturepeptide is located between the signal peptide and precursor-relatedpeptide in AKH/RPCH precursors, and at the C-terminal end inthe PDH/PDF precursors. The precursor-related (associated) peptidesin RPCH and PDH precursors in Crustacea show little or no similarityto corresponding domains of AKH and PDF precursors in insects.Although the functions of precursor-related peptides are unknown,the mature peptides are shown to serve diverse functions. RPCH'sactions in crustaceans include: pigment concentration in oneor more types of chromatophores, dark-adaptational screeningpigment movement in distal eye pigment cells, increase of retinalsensitivity, and neuromodulation. The related AKHs largely influencemetabolism in insects, although they serve additional functions.PDHs trigger pigment dispersion in chromatophores and inducelight-adaptational screening pigment movements in extraretinulareye pigment cells. The related PDFs appear to serve as a transmitterof circadian signals in the regulation of biological rhythmsin insects. Evolutionary relationships among the PDH/PDF peptidesand directions for future research are discussed.     

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.     

Novel adipokinetic hormones in the kissing bugs Rhodnius prolixus,Triatoma infestans,Dipetalogaster maxima and Panstrongylus megistus

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.     

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry

Amino acid sequences have been assigned to two cockroach neuropeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, M I, and Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.     

Preliminary characterization of enzyme activities in Malpighian tubules involved in the breakdown of adipokinetic hormones

Adipokinetic hormones (AKH) from different insect species, crustacean red pigment-concentrating hormone (RPCH), and synthetic substrates were used to characterized enzyme activities present in the Malpighian tubules (MT) of the desert locuts, Schistocerca gregaria, which are involved in the degradation of AKH. When peptides containing proline (position 6) were incubated with MT homogenate they were cleaved by a post-proline cleaving enzyme (PPCE). The presence of such an enzyme was confirmed by the breakdown of a synthetic substrate for PPCE. Peptides which do not contain proline were broken down by a post-phenylalanine cleaving enzyme (PFCE) which could be chymotrypsin or chymotryptic. This PFCE activity(ies) seem(s) to be inactive on the proline-containig peptides or their fragments or digests these at a slow rate. The C-terminal chymotrypsin fragments of the AKHs were broken down by MT homogenates with no accumulation of new intermediate products. It is not clear whether another endopeptidase, PPCE, or leucine aminopeptidase (LAP) is responsible. The MTs contain LAP activity; however, this enzyme(s) may be different from its vertebrate counterpart(s). Homogenates of MTs break down equimolar amounts of Pro-7AMC at approximately the same rate, while porcine kidney LAP (cytosol) cleaved Pro-7AMC much slower than Leu-7AMC. The demonstration of carboxypeptidase (CP) A and B activity in the MTs was not possible using conventional substrates such as hippuryl derivatives of amino acids. When CPA from porcine pancreas was added to MT homogenates hippuryl-phenylalanine was digested proving that the conditions were appropriate for CPA activity to occur. The treatment of a N-terminally blocked peptide fragment with MT homogenate led to the breakdown of the peptide giving evidence that the MT CP requires a substrate with a somewhat longer length of amino acid residues.     

Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.