Regulatory mechanisms underlying pheromone biosynthesis activating neuropeptide (PBAN)-induced internalization of the Bombyx mori PBAN receptor

Internalization of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor (PBANR) has been attributed to the presence of a 67 amino acid C-terminal extension absent in PBANRs from Helicoverpa. To identify the structural motif(s) responsible for internalization, a series of truncation mutants fused with enhanced green fluorescent protein were constructed and transiently expressed in insect Sf9 cells. Confocal microscopy analyses revealed that truncation at Gly357 severely inhibited internalization while truncation at Gln367 did not, indicating that the PBANR internalization motif resides between Gly357-Gln367. Alanine substitution studies suggest that Tyr360 and Leu363 may constitute a YXXL endosomal targeting motif that facilitates endocytosis, however, this motif does not appear to be the primary determinant; an indication that multiple sites are involved. Furthermore, we determined that internalization of the PBANR proceeds via a clathrin-dependent pathway, is dependent on the influx of extracellular calcium, and likely does not involve a G protein-coupled receptor kinase.     

Comparative neuroanatomy and genomics of hugin and pheromone biosynthesis activating neuropeptide (PBAN)

The Drosophila hugin gene encodes a prepropeptide that can potentially generate several neuropeptides.(1) The gene is expressed in 20 cells of the subesophageal ganglion (SOG) that are involved in modulating feeding behavior.(2) One of the hugin neuropeptides shares homology with mammalian neuromedin U8 (NmU8), which has been shown to regulate feeding behavior in rodents.(3,4) Recent clonal analysis indicated that each hugin expressing neuron projects to one of four main targets: the protocerebrum, the ventral nerve cord, the pharynx and the corpora cardiaca.(5) In addition all hugin neurons send short neurites to a novel region ventro-lateral to the foramen, which we suggested could be the tritocerebrum. In this short article, we discuss two specific issues brought up by these analyses. One concerns the polarity of hugin neurons. The other is an evolutionary perspective on the processing of hugin neuropeptides in light of new data from mass spectrometric and genomic analyses.     

A cDNA, from Agrotis ipsilon, that encodes the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides.

A cDNA encoding the prohormone of the pheromone biosynthesis activating neuropeptide (PBAN) in the moth Agrotis ipsilon was isolated. The cDNA contains 834 nucleotides, coding for a 193-amino acid protein that exhibits 89% identity with PBAN prohormones of other moths. The prohormone contains five potential peptides belonging to the FXPRL family. The peptide corresponding to the Bombyx mori diapause hormone exhibits an extra residue, and the C-terminal leucine is replaced by an isoleucine, introducing a new type of variability in this family of peptides. Northern blot analysis revealed expression in suboesophagal ganglion complexes. Constitutive heterologous expression of Agi-PBAN cDNA in yeast, using three different antibodies, did not produce PBAN-immunoreactive material.     

Backbone cyclic pheromone biosynthesis activating neuropeptide (PBAN) antagonists: inhibition of melanization in the moth Spodoptera littoralis (Insecta, Lepidoptera)

Antagonistic and agonistic activities of backbone cyclic (BBC) pheromone biosynthesis activating neuropeptide (PBAN) analogues were evaluated in an attempt to identify potent melanotropic antagonists, to gain an insight into their structure-activity relationship (SAR), and to discover molecules with selective and non-selective melanotropic and pheromonotropic properties. Eight potent melanotropic BBC antagonists and seven agonists were disclosed. SAR studies revealed that the structural requirements of the melanotropic and pheromonotropic agonists and antagonists are different. The cyclic structure of the BBC peptides was unimportant for antagonistic activity, and linearization retained their melanotropic and pheromonotropic antagonistic properties. Comparison of the antagonistic activities of the BBC and precyclic peptides with respect to both functions revealed eight selective antagonists (six that were selective melanotropic antagonists and two selective pheromonotropic antagonists) and four non-selective (melanotropic and pheromonotropic) antagonists. The selective melanotropic antagonists exhibited both, pure or mixed agonistic/antagonistic activities. The selective pheromonotropic compounds were pure antagonists. All non-selective compounds were pure antagonists. Comparison of the agonistic activities of the BBC peptides with respect to both functions revealed six selective melanotropic agonists and one non-selective agonistic compound. All compounds (whether selective or non-selective) exhibited pure agonistic activity. Discovery of the selective compounds hints at the possibility that the receptors that mediate the respective activities may have different properties.     

The distribution of PBAN (pheromone biosynthesis activating neuropeptide)-like immunoreactivity in the nervous system of the gypsy moth,Lymantria dispar

The production of sex pheromone in many moths is regulated by the neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). Studies in a number of species have shown that pheromone production can be linked to a hemolymph factor and that continuity in the ventral chain of ganglia is not required. However, it has recently been shown that production of pheromone in the gypsy moth, Lymantria dispar, is largely prevented in females with a transected ventral nerve cord (VNC). To begin to understand the cellular basis for this dependence on the VNC, we sought to determine the distribution of PBAN in the central nervous system and its neurohemal sites, including those associated with the VNC. Using an antiserum to L. dispar-PBAN in immunocytochemical methods, we have mapped the distribution of PBAN-like immunoreactivity (PLI). PLI is found in three clusters of ventral midline somata in the subesophageal ganglion (SEG), in three clusters of midline cells in each segmental ganglion, and in bilateral pairs of cells located posterolaterally in each abdominal ganglion. The SEG cells comprise both interneurons, with endings in the neuropil of each segmental ganglion, as well as neurosecretory cells, with endings in the retrocerebral complex and in an unusual neurohemal structure near the anterior aspect of the SEG. The latter structure, which we have named the corpus ventralis, receives axons from the two anterior clusters of cells in the SEG. In the abdominal ganglia, the posterolateral clusters of cells have immunoretroreactive axons exiting the ganglia via the ventral nerves. Endings of these axons reach the perivisceral organ in the next posterior ganglion and pass anteriorly into the median nerve, forming additional varicose endings. We did not detect PLI in the terminal nerve. Thus, our findings raise the possibility that the requirement for an intact VNC in pheromone production reflects a role for descending regulation of neurosecretory cells in the segmental ganglia. Arch. Insect Biochem. Physiol. 34:391–408, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.     

The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: identification, functional expression, and structure-activity relationships of ligand analogs

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>P4>T3>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [(3)H]tyrosyl-PBAN28-33NH(2) to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO(3)(-) ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K(d) of 5.73 +/- 1.05 x 10(-6) M and a Bmax of 1.85 +/- 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH(2) and PBAN28-33NuEta(2) with a K(i) of 4.3 +/- 1.1 x 10(-6) M and 4.9 +/- 2.6 x 10(-6) M, respectively.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands

The control of Spodoptera littoralis sex pheromone biosynthesis has been investigated with synthetic pheromone biosynthesis activating neuropeptide (PBAN) and different labeled tracers using an in vitro isolated gland system. Responsiveness of the glands to PBAN stimulation was impaired by careless tissue manipulation. The fact that PBAN is active in the isolated gland system suggests that this might be a target organ for this peptide in S. littoralis. As reported previously with Br-SOG extracts and intact females, label incorporation into the pheromone increased in glands treated with PBAN from all the precursors tested. However, the formation of labeled intermediates from d₅E11–14:Acid also occurred in glands incubated in the absence of the peptide, but the amounts of d₅Z9, E11–14:Acid were lower in PBAN treated glands than in controls. These results indicate that PBAN controls pheromone biosynthesis in S. littoralis by regulating the reduction of acyl moieties. © 1994 Wiley-Liss, Inc.     

Molecular modeling of the binding of pheromone biosynthesis activating neuropeptide to its receptor

Moth sex-pheromone biosynthesis follows a circadian cycle, which is cued by the release of the neurohormone pheromone biosynthesis activating neuropeptide (PBAN) to the hemolymph. PBAN binds to a G protein-coupled receptor (GPCR), in pheromone glands, (PG) initially identified by us in Helicoverpa zea moths (HezPBAN-R). In this study, the sequences of the seven transmembrane helices of HezPBAN-R were identified, built, packed and oriented correctly after multiple sequence alignment of the HezPBAN-R and several other GPCRs using the X-ray structure of rhodopsin as a template. Molecular dynamics simulations were run on three different beta-turn types of the C-terminal hexapeptide of PBAN and the results clustered into 12 structurally distinct groups. The lowest energy conformation from each group was used for computer-simulated docking with the model of the HezPBAN-R. Highest scoring complexes were examined and putative binding sites were identified. Experimental studies, using in vitro PG, revealed lower levels of pheromonotropic activity when challenged with pyrokinin-like peptides than with HezPBAN as ligand. Thus, the Drosophila melanogaster pyrokinin-1 receptor (CG9918) was chosen to create chimera receptors by exchanging between the three extracellular loops of the HezPBAN-R and the CG9918 for in silico mutagenesis experiments. The predicted docking model was validated with experimental data obtained from expressed chimera receptors in Sf9 cells.     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide

(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by Delta(11)-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluorobenzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass spectrometry is also reported.     

Synthesis and biological activity of a photoaffinity-biotinylated pheromone-biosynthesis activating neuropeptide (PBAN) analog.

To study the mode of action of pheromone-biosynthesis activating neuropeptide (PBAN) at the receptor level and for receptor purification, we synthesized and tested the biologic properties of a photoaffinity biotinylated PBAN analog N-[N-(4-azido-tetrafluorobenzoyl)-biocytinyloxyl-succinimide (Atf-Bct-NHS-PBAN). The Atf-Bct-NHS-PBAN was separated from unreacted reagent and synthetic Hez-PBAN by high-performance liquid chromatography. Conjugated biotin was detected by using enzyme-linked assay as well as tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis. The biologic activity of purified Atf-Bct-NHS-PBAN was confirmed using both in vivo and in vitro pheromonotropic bioassays. These observations indicate that Atf-Bct-NHS-PBAN is a full agonist of PBAN action in pheromone glands and may be used to study PBAN receptors by employing avidin coupled to various reporter groups.     

Tissue localization and partial characterization of pheromone biosynthesis activating neuropeptide inAchaea janata

Female sex pheromone production in certain moth species have been shown to be regulated by a cephalic endocrine peptidic factor: pheromone biosynthesis activating neuropeptide (PBAN), having 33 amino acid residues. Antisera against syntheticHeliothis zea-PBAN were developed. Using these polyclonals, immunoreactivity was mapped in the nervous system ofAchaea janata. Three distinct groups of immunopositive secretory neurons were identified in the suboesophageal ganglion; and immunoreactivity was observed in the corpora cardiaca, thoracic and in the abdominal ganglia. From about 6000 brain sub-oesophageal ganglion complexes, the neuropeptide was isolated; and purified sequentially by Sep-pak and reversed phase high performance liquid chromatographic methods. Identity of purified PBAN fraction was confirmed with polyclonal antibody by immunoblotting. Molecular mass of the isolated peptide was determined by matrix-assisted laser desorption/ionization mass spectrometry, and was found to be 3900 Da, same as that of knownH. zea-PBAN. Radiochemical bioassay confirmed the pheromonotropic effect of the isolated neuropeptide in this insect     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。