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.     

Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera).

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.     

The newly discovered insect order Mantophasmatodea contains a novel member of the adipokinetic hormone family of peptides

A novel member of the AKH/RPCH family of peptides has been identified from the corpus cardiacum of an, as yet, unidentified species of the newly discovered insect order Mantophasmatodea from Namibia. The primary sequence of the peptide, which is denoted Manto-CC, was deduced from multiple MS(N) electrospray mass data to be an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide. Synthetic Manto-CC co-elutes on reversed-phase HPLC with the natural peptide from the gland of the insect. Interestingly, Manto-CC is structurally very closely related (only one point mutation) to the AKH/RPCH peptides previously identified in mostly more basal insect taxa (Odonata, Blattodea, and Ensifera) and in Crustacea, the sister group of insects, whereas larger structural differences occur with peptides from Mantodea and Phasmatodea, which are thought to be close relatives of Mantophasmatodea. Functionally, Manto-CC may be employed to activate glycogen phosphorylase to mobilize carbohydrates.     

Molecular and functional characterization of adipokinetic hormone receptor and its peptide ligands in Bombyx mori

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides, but its signaling pathways remain to be elucidated. In this study, we molecularly characterized the signaling of Bombyx AKH receptor (AKHR) and its peptide ligands in HEK293 cells. In HEK293 cells stably expressing AKHR, AKH1 stimulation not only led to a ligand concentration dependent mobilization of intracellular Ca²⁺ and cAMP accumulation, but also elicited transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We observed that AKH receptor was rapidly internalized after AKH1 stimulation. We further demonstrated that AKH2 exhibited high activities in cAMP accumulation and ERK1/2 activation on AKHR comparable to AKH1, whereas AKH3 was much less effective.     

Structure-activity relationships on hyperglycemia by representatives of the adipokinetic/hyperglycemic hormone family in Blaberus discoidalis cockroaches

Summary Several members of the adipokinetic/hyperglycemic neurohormone family from several different invertebrate species have been prepared by solid-phase peptide synthesis and assayed by a modified in vivo hyperglycemic bioassay in Blaberus discoidalis cockroaches. The hypertrehalosemic hormone (HrTH) is the endogenous hypertrehalosemic factor for B. discoidalis and was the most potent peptide in the assay. The more divergent the sequence of a family member from Blaberus HrTH, the less potent was the bioanalog. Manduca adipokinetic hormone is the most divergent peptide of the family and was totally inactive in the bioassay. Locusta adipokinetic hormone I had reduced maximum activity in the assay, which suggests that Ser⁵ is an important residue for the transduction of the hyperglycemic response. The direct relation between bioanalog similarity to Blaberus HrTH sequence and potency suggests that the hormone and target cell receptor for HrTH have evolved to maintain an optimal fit.Abbreviations AKH adipokinetic hormone - HrGH hyperglycemic hormone - HrTH hypertrehalosemic hormone - RPCH red pigmentconcentrating hormone - CAH cardioacceleratory hormone. Hormone abbreviations are according to the convention of Gäde and Raina (1989) except that the genus names are not abbreviated     

Isolation and characterization of red-pigment-concentrating hormone (RPCH) from six crustacean species

Summary Red-pigment-concentrating hormone (RPCH) has been isolated from nerve tissue of six decapod crustaccan species. The primary structure of three of the six hormones, i.e., those ofCancer magister, Carcinus maenas andOrconectes limosus, was determined by manual microsequencing as: pELNFSPGW-NH₂. This sequence is identical to that of RPCH fromPandalus borealis, the only previously known sequence of a crustacean RPCH. The other three hormones fromLiocarcinus puber, Nephrops norvegicus, andPacifastacus leniusculus could not be characterized completely. However, amino acid compositions, the presence of N-terminal pGlu, and the blocked N-terminal ends are in accordance with the primary structure established for the other three RPCHs. We suggest that all six peptides have the same amino acid sequence. These results indicate that RPCH, which is likely to be related to the peptides of the AKH family in insects, is highly conserved among crustacean species. This is in remarkable contrast to the high degree of molecular evolution exemplified by the many different AKH-like peptides among insect species.Abbreviations AKH adipokinetic hormone - Aufs absorption units full scale - BSA bovine serum albumin - ECH erythrophore concentrating hormone (=RPCH) - EDTA ethylenediamine-tetra-acetic acid - HOAc acetic acid - HPLC high pressure liquid chromatography - O.D. optical density - PDH pigment dispersing hormone - PGAP pyroglutamate aminopeptidase - RPCH red-pigment-concentrating hormone (=ECH) - SOG suboesophageal ganglion - TFA trifluoroacetic acid     

Degradation of adipokinetic hormone family peptides by a circulating endopeptidase in the insect Manduca sexta.

The hemolymph (blood) of the Lepidopteran insect Manduca sexta contains an endopeptidase that metabolizes the nonapeptide Manduca adipokinetic hormone. In contrast to the situation in other insects, where the major site of inactivation is the Malpighian tubules (excretory organs), in Manduca the capacity of the hemolymph to metabolize adipokinetic hormone is comparable to that of the Malpighian tubules. The hemolymph enzyme cleaves Manduca adipokinetic hormone (pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2) to give the fragment pGlu-Leu-Thr-Phe-Thr. Other fragments were not positively identified. The enzyme is present in the plasma and not in hemocytes, and occurs at similar levels in the hemolymph of larvae, pupae and adults. The enzyme is inactivated by boiling, has a neutral pH optimum (7.0-7.5), and an estimated molecular weight of 66 kDa. The enzyme was strongly inhibited by inhibitors of metalloprotease activity (EGTA and 1,10-phenanthroline), but not by serine protease inhibitors. The enzyme was capable of metabolizing a number of AKH family peptides with varying sequences around the presumed site of cleavage. An accurate assessment of enzyme kinetics was not possible with the assay method used, but the enzyme was not saturated at a substrate concentration of 10 microM, and the value of Km must be at least 1 microM. It is possible that the enzyme may represent a low affinity system of peptide removal rather than the principal means of inactivation.     

Open conformation of adipokinetic hormone receptor from the malaria mosquito facilitates hormone binding

Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a ‘closed’ structure while the beta2-based structure is ‘open’. The ‘open’ conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH₂) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH₂) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.     

AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum

An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.     

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.     

The first identified neuropeptide in the insect order Megaloptera: a novel member of the adipokinetic hormone family in the alderfly Sialis lutaria

This is the first report on the structural identity of a neuropeptide of the insect order Megaloptera. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca glands of the alderfly, Sialis lutaria. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Thr-Phe-Thr-Pro-Ser-Trp amide. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by comparing retention time on reversed-phase HPLC and establishing co-elution with the synthetic Leu(2)-form which also had exactly the same MS(2) mass spectra as the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which has already more than 40 members. Interestingly, the primary structure is identical to that predicted from genome information for the adipokinetic hormone of the yellow fever mosquito, Aedes aegypti. Since alderflies are not known for their active flight metabolism but produce a rather high number of eggs, it is anticipated that the alderfly is a good study object to establish a possible role of the novel peptide to regulate fat mobilization from the fat body and transport into the egg, thereby playing a role in the control of reproductive processes.     

Preliminary characterization of glycogen phosphorylase activating hormone and adipokinetic hormone from Manduca sexta corpora cardiaca

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G-25 and from Sephadex LH-20 columns. In the separation experiments with glass beads and G-25 the activities eluted as a single peak, but using LH-20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × V_t, which is very similar to locust AKH, while the smaller second peak eluted at O.74 × V _t. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.     

Synthesis and biological activity of adipokinetic hormone analogues modified at the C-terminus

A series of Locusta adipokinetic hormone I (AKH-I), C-terminal threonine residue using a combination of solid- and liquid-phase methodology and evaluated in Locusta migratoria, in a lipid mobilization assay in vivo and an acetate uptake assay in vitro. Modifications at Thr¹⁰ of AKH-I involved replacement of its C-terminal amide by the groups -OH, -OCH₃, -NHCH₃, -N(CH₃)₂, and -NHC₆H₅; the last three groups were also applied to the amide of AKH-I-[Thr(Brl)¹⁰]. The methyl ester, monomethyl, and dimethyl analogues were all of lower activity than the parent in the lipid mobilization assay, but lost less than two orders of potency. In the acetate uptake assay, again the methyl ester analogue showed the greatest retention of biological activity of all modified peptides. A cyclic analogue, cyclo (PLNFTPNWGT), was active in both assay, but only at very high concentrations. Almost all analogues were more active in the acetate uptake assay than in the lipid assay, but unusually, AKH-I-NHCH₃ and AKH-I-N(CH₃)₂, together with cyclo (PLNFTPNWGT), were more active in the lipid mobilization assay. In addition, the acid AKH-I analogue did not suffer as large a loss in potency in the lipid mobilization assay as in the acetate uptake assay, although it was less potent in the former. The relative potencies of these two methyl analogues contrast with those for AKH AKH-I-[Thr(Bzl)¹⁰]-NHCH₃ and AKH-I-[Thr(Bzl)¹⁰]-N(CH₃)₂, which, together with both phenyl analogues, were significantly more active in the acetate uptake assay. We conclude that the acetate uptake assay has a greater preference for a hydrophobic C-terminus, compared with the lipid mobilization assay.     

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.     

The N-terminal domain of betaB2-crystallin resembles the putative ancestral homodimer

betagamma-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains reflecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the betagamma-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 A X-ray structure of the homodimer of the N-terminal domain of rat betaB2-crystallin (betaB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native betagamma-crystallins, where N and C-terminal domains (which share approximately 35% sequence identity) are related by a pseudo-2-fold axis. betaB2-N thus resembles the ancestral prototype of the betagamma-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in betagamma-crystallins, but without the benefit of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the betagamma-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.     

Insect diapause-specific peptide from the leaf beetle has consensus with a putative iridovirus peptide

Diapause and hibernation during periods of environmental adversity are essential features of the life cycle in many organisms, yet the molecular basis for these events differs among animals. We have identified an endogenous diapause/hibernation-specific peptide, from the leaf beetle Gastrophysa atrocyanea. This peptide provides antifungal activity, acts as a N-type voltage-gated Ca2+ channel blocker, and has a new consensus sequence with an unknown polypeptide encoded in the insect iridescent virus. These results indicate that the diapause-specific peptide may be utilized as a probe to analyze and compare functional and evolutional aspects of the life cycles of insects and iridoviruses.     

The role of adipokinetic hormone in the control of vitellogenesis in locusts

Vitellogenesis in locusts is synchronized with the cyclic maturation of oocytes. Vitellogenesis by excised fat body of gravid females is differentially inhibited 80–90% when locust adipokinetic hormone I (AKH-I) is added to the incubation media. Hemolymph methanolic extracts completely inhibit fat body protein synthesis in vitro when the donor females are at the end of the ovarian cycle (6 mm stage), but not when taken from earlier stages. Hemolymph methanolic extracts from vitellogenic females at the 6 mm stage are separated by HPLC into three distinct inhibitors of protein synthesis, one of which is AKH-I. AKH-RIA of hemolymph during the first ovarian cycle reveals no AKH-I during active vitellogenesis, but a marked increase to about 5 ng per female at the end of egg maturation. A development of responsiveness to AKH-I is evident in female fat body as vitellogenesis proceeds. AKH-I is involved in the negative control of vitellogenesis.