A pheromonotropic peptide of Helicoverpa zea,with melanizing activity,interaction with PBAN,and distribution of immunoreactivity

The sequence of an 18-amino acid residue peptide was deduced from the gene encoding PBAN and other peptides with common C-termini in Helicoverpa zea. The peptide caused melanization in larvae and pheromone production in females of H. zea, and was designated pheromonotropic melanizing peptide (Hez-PMP). The peptide has a 83% sequence homology with a pheromonotropic peptide isolated from Pseudaletia separata. PMP caused melanization and mortality when injected into larvae just before molting. Whereas intense melanization was caused with a dose of 1,000 pmol, peak mortality occurred at 100 pmol, with 50% of larvae dying within 48 h after injection. Pheromonotropic activity of PMP was dose dependent. Co-injection of Hez-PMP and Hez-PBAN into a female resulted in suppression of the pheromonotropic effect of PBAN. Whole-mount immunocytochemical studies revealed PMP-like immunoreactivity in frontal ganglion, subesophageal, thoracic, and abdominal ganglia as well as the esophageal nerve.     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.)

The correlation between triacylglycerols containing conjugated diene fatty acyl moieties and pheromone aldehydes in the sex pheromone glands of females of Manduca sexta was investigated. Females decapitated 15 h after adult emergence neither called nor produced pheromone during the natural period of pheromone production on the subsequent two nights. However, these females could be stimulated to produce sex pheromone for prolonged periods by repeated injection of synthetic pheromone biosynthesis activating neuropeptide (PBAN). Gas chromatographic analysis of methanolysis products of lipids extracted from the pheromone glands of decapitated and intact females showed no differences in the amounts of fatty acyl precursors of pheromone. High performance liquid chromatographic analysis of the triacylglycerols containing conjugated diene analogues of the pheromone components (diene TG), obtained 24 and 48 h after decapitation, showed that the total amounts of these components were not affected by decapitation. The amounts of all diene TG peaks declined significantly when decapitated females were stimulated to produce pheromone during a 7 h period by repeated injection of PBAN at 3 h intervals but recovered when pheromone production subsided. These results indicate that PBAN induces liberation of pheromone precursors from the triacylglycerols during pheromone biosynthesis but does not induce replenishment of this storage pool. © 1996 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

     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 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.

     

中国野桑蚕性信息素合成激活肽(PBAN)基因的克隆及序列分析

根据家蚕(Bombyx mori)性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)基因DNA序列设计引物,扩增获得中国野桑蚕(Bombyx mandarina China)PBAN基因。分析表明,PBAN由33个氨基酸组成,在第14个氨基酸异亮氨酸和第15个氨基酸酪氨酸之间插入了698bp的内含子。根据PBAN及其基因cDNA、DNA序列分别构建分子进化树,结果显示3个水平比对结果构建的分子进化树有较好的一致性,推测PBAN基因可能适合于科、属之间的进化分析;并且PBAN基因内含子没有表现出特有的进化信息,推测PBAN基因内含子的进化与PBAN全长基因的进化在进化速率上并没有显著差别。相对于PBAN及α—SGNP、γ—SGNP,β—SGNP的进化速率相对较快,推测β—SGNP序列可能适合用于种间的进化分析。     

CONTROL OF SEX PHEROMONE BIOSYNTHETIC PATHWAY BY PBAN IN ASIAN CORN BORER OSTRINIA FURNACALlS*

Abstract Sex pheromone titer in Ostrinia furnacalis was significantly decreased to a very low level by decapitation, but it could be restored by injection of head extract prepared from both male and female moths or synthetic pheromone biosynthesis activating neuropepide (PBAN). This fact indicates that pheromone production is under the control of a PBAN-like factor. The sex pheromone biosynthetic pathway of O. furnacalis originates with the biosynthesis of palmitic acid and followed by A14 desaturation, chain shortening, reduction and acetylation to form the pheromone components, (Z) and (E)-12-tetradecenyl acetate. In order to determine which step in the pathway is controlled by PBAN, the incorporation of different labeled precursors into the pheromone and its intermediate were studied. Our results suggest that PBAN controls pheromone biosynthesis in O. furnacalis by mainly regulating an early step from acetate to palmitic acid.     

PBAN对亚洲玉米螟(Ostrinia furnacalis)性外激素生物合成路线的控制(英)

去头处理致使亚洲玉米螟性外激素的含量显著地下降到很低的水平。注射雄性或雌性玉米螟头部提取物或合成PBAN(激活外激素生物合成神经肽)可使外激素的含量得以恢复。因此可知玉米螟外激素的产生系受一种类PBAN因子控制。玉米螟性外激素生物合成路线由棕榈酸的生物合成开始,然后经14位脱饱和化,碳链缩短,还原和乙酰化形成外激素顺和反12—十四碳烯乙酸酯。为了阐明受PBAN控制的生物合成步骤,研究了不同的标记前体掺入外激素及其中间体的情况。根据结果推论,PBAN主要通过调节由乙酸酯到棕榈酸的生物合成步骤来控制外激素生物合成。     

Regulation of sex pheromone biosynthesis in two noctuid species,S. littoralis and M. brassicae,may involve both PBAN and the ventral nerve cord

In order to understand better the mechanism of regulation of pheromone production in moth species, we performed ELISA analyses to detect and follow pheromone biosynthesis activating neuropeptide-like immunoreactivity (PBAN-IR) in different tissues of the two noctuidae species, Spodoptera littoralis and Mamestra brassicae. Male S. littoralis and both male and female M. brassicae brain-subesophageal ganglion (Br-SEG), corpora cardiaca-corpora allata complex, and terminal abdominal ganglion extracts showed the presence of PBAN-IR during both the photophase and the scotophase. However, PBAN-IR was found only in scotophase in female hemolymph. Analysis of extracts of Br-SEG, terminal abdominal ganglion, and hemolymph after HPLC fractionation showed that the most immunoreactive fraction in all the extracts exhibited the same retention time as Hez-PBAN, suggesting that similar PBAN-like material is present in all these tissues. In vivo studies demonstrated that severing the ventral nerve cord in M. brassicae anterior to the terminal abdominal ganglion impaired normal sex pheromone production by third-scotophase females, as was previously shown in S. littoralis. Additionally, PBAN-IR levels were lower in hemolymph samples obtained at the peak of pheromone production in both S. littoralis and M. brassicae females that had the ventral nerve cord severed compared with sham operated animals. These results, along with earlier reported data, indicate that control of pheromone production in both species may involve both PBAN (or PBAN-like peptides) and the ventral nerve cord and support the hypothesis that a neural input from the ventral nerve cord triggers the release of the pheromonotropic peptide(s) into the hemolymph, which then acts directly on the pheromone gland to stimulate pheromone biosynthesis. Arch. Insect Biochem. Physiol. 37:295–304, 1998. © 1998 Wiley-Liss, Inc.

1 We thank Germán Lázaro for insect rearing.

     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the C-terminal.Its effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a reporter.Secretion of EGFP fusion proteinswas examined under fluorescence microscope.FibH SPs with lengths of 20,18,16 and 12 a.a.can directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP.When 16a.a.of the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk gland.The cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.     

Isolation and Structure of staph-cAM373 Produced by Staphylococcus aureus That Induces Conjugal Transfer of Enterococcus faecalis Plasmid pAM373

Enterococus faecalis plasmid pAM373 encodes a mating response to the sex pheromone, cAM373, which is secreted from pAM373-free E. faecalis. cAM373-like activity was detected in a culture filtrate of Staphylococcus aureus. The major active substance, termed staph-cAM313 was isolated, and its structure was identified as a H-Ala-Ile-Phe-Ile-Leu-Ala-Ala-OH heptapeptide.     

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。     

Opioid activity of synthetic deltorphin II analogues and their spin labeled derivatives

Summary Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly, NH₂, Del II), an endogenous linear heptapeptide, is a highly selective agonist of the δ-opioid receptor. To study the effect of the position 4 residue (Glu) on the opioid activity of Del II, we designed and synthesized three analogues of Del II by solid-phase peptide synthesis. They were [Val⁴, Glu⁵]Del II, [Val⁴, Glu⁶]Del II and [Gly⁴, Glu⁷]Del II. To study the effect of spin labeling on peptide bioactivities, all the peptides were labeled using a free radical. The labeling material was a stable nitrogen-oxygen free radical which was linked to the N-terminal via an amide bond. We investigated the opioid bioactivities of these analogues both in vivo and in vitro, and concluded that the differences in opioid activity of Del II and its analogues were due to structural differences. When the Glu residue is at position 5 or 6, the internal hydrogen bonds in Del II are affected and there is a change in three-dimensional structure and opioid activity. The antinociceptive activity of all the peptides decreased after spin labeling. This indicates that the stable nitrogen-oxygen free radical is a dual-function spin-labeling molecule.     

烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.     

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.     

PBAN及其对昆虫性外激素的调控

本文综述了性外激素生物合成激活神经肽 (PBAN)及其对昆虫尤其是鳞翅目昆虫性外激素产生的调控 ,包括PBAN的结构、产生、转运、作用方式及保幼激素和蜕皮激素对性外激素合成的作用 ,并展望了未来的研究方向。