昆虫感觉气味的细胞与分子机制研究进展

昆虫作为地球上最为成功的类群,已经成功地进化了精细的化学感受系统,通过化学感受系统适应各种复杂的环境,保持种群的繁荣。自1991年在动物中发现嗅觉受体基因以来,关于昆虫感受化学信息的周缘神经系统的分子和细胞机制方面的进展十分迅速。文章主要就昆虫周缘神经系统的感受化学信息的分子和细胞机制进行综述。首先对昆虫感觉气味的细胞机制的研究进展进行简要介绍。昆虫嗅觉神经元在感受化学信息过程中起着极为重要的作用,昆虫嗅觉神经元上表达的嗅觉受体不同而执行着各异的功能。各种嗅觉神经元对于化学信息的感受谱有较大的区别;嗅觉神经元对化学信息类型、浓度、流动动态等产生相应的电生理特征反应。研究表明同一种神经原可以感受多种化学信息,而一种化学信息也可以被多种神经原所感受。由神经原对化学信息感受所形成的特征组合就是感受化学信息的编码。其次较为详细地论述与昆虫感受气味分子相关的一些蛋白质的研究进展。气味分子结合蛋白是一类分子量较小、水溶性的蛋白,主要位于化学感受器神经原树突周围的淋巴液中。在结构上的主要特征是具有6个保守的半光氨酸和由6个α螺旋组成的结合腔。自1981年发现以来,已经在40余种昆虫中发现上百种。由于研究手段的不断进步,已经对该类蛋白的表达特征、结合特性以及三维结构和结合位点进行了大量的研究,提出了多个可能的功能假说,在诸多的假说中,较为广泛接受的是气味分子结合蛋白在昆虫感觉气味的过程中,是与疏水性的气味分子相结合,并将气味分子运输到嗅觉神经原树突膜上的嗅觉受体上。这些处于树突膜上的嗅觉受体则是昆虫感觉气味过程中的另一个十分重要的蛋白质。目前,已经在果蝇、按蚊、蜜蜂和家蚕等10余个昆虫种类中发现上百个嗅觉受体蛋白基因。这类蛋白是跨膜蛋白,一般具有7个跨膜区,整个蛋白的氨基酸残基在400～600个。昆虫的嗅觉受体蛋白的N-端在胞内,而C-端在胞外,这与G耦联蛋白不同。而且,昆虫的一个嗅觉神经元可以表达1～3个嗅觉受体蛋白,也与哺乳动物的一个神经元只表达一种受体蛋白有所不同。每种嗅觉受体可以感受多种气味分子,而一种气味分子可以被多个嗅觉受体所感知,这样组成了感受化学信息的编码谱。最近采用基因敲除技术和膜片钳技术研究发现,昆虫的嗅觉受体蛋白在信号传导中也有特殊性,即嗅觉受体可以直接作为离子通道,而引起动作电位。还有近来的研究表明,神经膜蛋白对于果蝇的性信息素感受神经元感受性信息素cVA是必要的。实际上,昆虫对于化学信息的感受和信号的转导,并不是上述蛋白单独起作用完成的,而是多种蛋白相互作用的结果。论文最后对该领域研究内容进行了展望。     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

昆虫非典型嗅觉受体Orco的功能和分子结构研究进展

嗅觉受体是参与昆虫嗅觉识别过程的一类重要蛋白。在昆虫的众多嗅觉受体中, 有一类受体明显不同于其他受体, 被称为Orco。该受体基因在不同昆虫种间高度保守, 且表达广泛。Orco在昆虫嗅觉识别过程中发挥关键作用。采用基因突变或RNAi等技术使Orco基因沉默后, 昆虫会出现严重的嗅觉缺陷, 但Orco本身不与气味配体结合, 它与传统嗅觉受体形成复合体Or-Orco, 促进传统嗅觉受体在神经元树突膜上的定位并维持其稳定性, 提高传统嗅觉受体对气味反应的效率。昆虫嗅觉受体的结构与脊椎动物的G蛋白偶联受体相似, 均有7个跨膜区, 但二者的膜拓扑结构相反, 昆虫嗅觉受体的N末端位于细胞质膜内, C末端在细胞质膜外, Orco与传统嗅觉受体通过保守的C末端区域相互作用形成一种新型的配体门控离子通道--Or-Orco复合体。阐明Orco在昆虫嗅觉识别中的功能机制, 可为开创基于昆虫嗅觉行为干扰的新的害虫防治措施提供基础。     

An overview of odorant-binding protein functions in insect peripheral olfactory reception

Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligand-binding test; then, we review current progress around the points cited above to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.     

Fluorescence competition assay for the assessment of green leaf volatiles and trans-β-farnesene bound to three odorant-binding proteins in the wheat aphid Sitobion avenae (Fabricius)

Odorant-binding proteins (OBPs) are important parts of insect olfactory systems, and sensitive olfaction is vital for phytophagous insects in host foraging. Electrophysiological studies are helpful in understanding olfactory sensing in Sitobion avenae (Hemiptera: Aphididae), but the functions of odorant-binding proteins in this insect are poorly understood. In this study, we used fluorescence competition assays to measure the binding specificities of SaveOBPs. The results showed that both SaveOBP2 and SaveOBP3 were superior to SaveOBP7 in binding green leaf volatiles. It was unexpected that SaveOBP7 bound trans-β-farnesene strongly, which was known as alarm pheromone of this species. Host volatiles were recognized much more easily by SaveOBP2, and the observed binding activity of SaveOBP2 equaled for tested green leaf volatiles. Our results imply that SaveOBP7 might play a more important role in aphid alarm pheromone discrimination.     

昆虫嗅觉相关蛋白的研究进展

嗅觉是昆虫产生行为的重要物质基础,阐明昆虫嗅觉机理有助于调控昆虫行为和进行害虫治理。近年来,许多与嗅觉相关的生物活性分子和相关基因的发现和克隆,对揭示嗅觉机理具有重要作用。作者针对近年来研究较多的气味结合蛋白、化学感受蛋白、气味受体、气味降解酶以及感觉神经元膜蛋白等,就其生化特性、表达部位、分子结构、生理功能等进行了综述。     

Ligand binding and homology modelling of insect odorant‐binding proteins

This review describes the main characteristics of odorant‐binding proteins (OBPs) for homology modelling and presents a summary of structure prediction studies on insect OBPs, along with the steps involved and some limitations and improvements. The technique involves a computing approach to model protein structures and is based on a comparison between a target (unknown structure) and one or more templates (experimentally determined structures). As targets for structure prediction, OBPs are considered to play a functional role for recognition, desorption, scavenging, protection and transportation of hydrophobic molecules (odourants) across an aqueous environment (lymph) to olfactory receptor neurones (ORNs) located in sensilla, the main olfactory units of insect antennae. Lepidopteran pheromone‐binding proteins, a subgroup of OBPs, are characterized by remarkable structural features, in which high sequence identities (approximately 30%) among these OBPs and a large number of available templates can facilitate the prediction of precise homology models. Approximately 30 studies have been performed on insect OBPs using homology modelling as a tool to predict their structures. Although some of the studies have assessed ligand‐binding affinity using structural information and biochemical measurements, few have performed docking and molecular dynamic (MD) simulations as a virtual method to predict best ligands. Docking and MD simulations are discussed in the context of discovery of novel semiochemicals (super‐ligands) using homology modelling to conceive further strategies in insect management.     

昆虫气味结合蛋白的研究进展

摘要: 昆虫主要依赖其复杂且灵敏的化学感受系统来识别并区分外界环境中的各种化学信号。嗅觉是负责嗅觉信号传导的感官方式,能够引起昆虫觅食、产卵、交配和躲避天敌等对生存和繁殖至关重要的行为反应。在嗅觉感知过程中,气味结合蛋白(odorant binding proteins, OBPs)最先与外界脂溶性化学物质相互作用,并将其转运至化学受体神经元上,激活树突膜表面分布的嗅觉受体(olfactory receptors, ORs),是嗅觉系统正常运行的必需蛋白。近年来,随着高通量测序和分子生物学技术的快速发展,越来越多的昆虫OBPs相继得以鉴定并开展功能研究。昆虫OBPs是一类可溶性的小分子蛋白,一般由6个α-螺旋构成一个稳定、紧密的疏水性结合腔,其构象变化因昆虫种类和配体结构不同而有所差异。OBPs的分布不受限于嗅觉器官,还在口器、足、中肠、腺体等非嗅觉组织中表达,具有嗅觉识别、味觉感受、营养物质转运、信息素合成与释放、组织发育与分化等生理功能。OBPs行使以上功能的共同特性为结合和溶解包括信息素组分、普通气味分子和非挥发性物质等的疏水性小分子物质。昆虫OBPs的稳定性和多功能性暗示其可广泛应用于害虫防治、生物传感器、分析化学、生态学等多个领域。本文对过去20多年来昆虫OBPs的相关研究进行综述,为进一步深入开展OBPs的功能研究提供理论参考。     

Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class

Agam (Anopheles gambiae) relies on its olfactory system to target human prey, leading eventually to the injection of Plasmodium falciparum, the malaria vector. OBPs (odorant-binding proteins) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. In the present study, we undertook a large-scale analysis of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the three-dimensional structures of several Agam OBPs, either alone or in complex with model compounds. In the present paper, we report the first three-dimensional structure of a member of the C-plus class of OBPs, AgamOBP47, which has a longer sequence than classical OBPs and contains six disulfide bridges. AgamOBP47 possesses a core of six α-helices and three disulfide bridges, similar to the classical OBP fold. Two extra loops and the N- and C-terminal extra segments contain two additional α-helices and are held in conformation by three disulfide bridges. They are located either side of the classical OBP core domain. The binding site of OBP47 is located between the core and the additional domains. Two crevices are observed on opposite sides of OBP47, which are joined together by a shallow channel of sufficient size to accommodate a model of the best-tested ligand. The binding sites of C-plus class OBPs therefore exhibit different characteristics, as compared with classical OBPs, which should lead to markedly diverse functional implications.     

Insights on pH-dependent conformational changes of mosquito odorant binding proteins by molecular dynamics simulations

Chemical recognition plays an important role for the survival and reproduction of many insect species. Odorant binding proteins (OBPs) are the primary components of the insect olfactory mechanism and have been documented to play an important role in the host-seeking mechanism of mosquitoes. They are “transport proteins” believed to transport odorant molecules from the external environment to their respective membrane targets, the olfactory receptors. The mechanism by which this transport occurs in mosquitoes remains a conundrum in this field. Nevertheless, OBPs have proved to be amenable to conformational changes mediated by a pH change in other insect species. In this paper, the effect of pH on the conformational flexibility of mosquito OBPs is assessed computationally using molecular dynamics simulations of a mosquito OBP “CquiOBP1” bound to its pheromone 3OG (PDB ID: 3OGN). Conformational twist of a loop, driven by a set of well-characterized changes in intramolecular interactions of the loop, is demonstrated. The concomitant (i) closure of what is believed to be the entrance of the binding pocket, (ii) expansion of what could be an exit site, and (iii) migration of the ligand towards this putative exit site provide preliminary insights into the mechanism of ligand binding and release of these proteins in mosquitoes. The correlation of our results with previous experimental observations based on NMR studies help us provide a cardinal illustration on one of the probable dynamics and mechanism by which certain mosquito OBPs could deliver their ligand to their membrane-bound receptors at specific pH conditions.     

Odorant binding protein diversity and distribution among the insect orders, as indicated by LAP, an OBP-related protein of the true bug Lygus lineolaris (Hemiptera, Heteroptera)

Insect odorant binding proteins (OBPs) are thought to deliver odors to olfactory receptors, and thus may be the first biochemical step in odor reception capable of some level of odor discrimination. OBPs have been identified from numerous species of several insect orders, including Lepidoptera, Diptera, Coleoptera and Hymenoptera; all are holometabolous insects belonging to the monophyletic division of insects known as the Endopterygota. Recently, an antennal protein with OBP-like properties was identified from Lygus lineolaris, a hemipteran insect representing the Hemipteroid Assemblage, a sister division to the Endopterygota. The full length sequence of Lygus antennal protein (LAP) is presented in this report. In situ hybridization analysis revealed LAP expression in cell clusters associating with olfactory sensilla; expression was adult-specific, initiating in developing adult tissue during the transitional period that precedes the actual adult molt. Sequence analysis confirmed that LAP is homologous with the OBP-related protein family, and most similar to the OS-E and OS-F proteins of Drosophila, the ABPX proteins of Lepidoptera and the OBPRP proteins of the Coleoptera. Assuming that the OBP-related proteins represent one homologous family, the identification of LAP significantly expands the phylogenetic depth of that family and its underlying role in odor detection to encompass all members of the Endopterygota and Hemipteroid Assemblage, which comprise >90% of all insect species.     

The crystal structure of odorant binding protein 7 from Anopheles gambiae exhibits an outstanding adaptability of its binding site

Anopheles gambiae (Agam) targets human and animals by using its olfactory system, leading to the spread of Plasmodium falciparum, the malaria vector. Odorant binding proteins (OBPs) participate to the first event in odorant recognition and constitute an interesting target for insect control. OBPs interact with olfactory receptors to which they deliver the odorant molecule. We have undertaken a large-scale study of proteins belonging to the olfactory system of Agam with in mind of designing strong olfactory repellants. Here, we report the expression, three-dimensional structures and binding properties of AgamOBP07, a member of a new structural class of OBPs, characterized by the occurrence of eight cysteines. We showed that AgamOBP07 possesses seven α-helices and four disulfide bridges, instead of six α-helices and three disulfide bridges in classical OBPs. The extra seventh helix is located at the surface of the protein, locked by the fourth disulfide bridge, and forms a wall of the internal cavity. The binding site of the protein is mainly hydrophobic, elongated and open and is able to accommodate elongated ligands, linear or polycyclic, as suggested also by binding experiments. An elongated electron density was observed in the internal cavity of the purified protein, belonging to a serendipitous ligand. The structure of AgamOBP07 in complex with an azo-bicyclic model compound reveals that a large conformational change in the protein has reshaped its binding site, provoking helix 4 unfolding and doubling of the cavity volume.     

昆虫嗅觉结合蛋白研究进展

嗅觉结合蛋白是嗅觉系统的第一个参与者,主要表达在嗅觉外周系统淋巴液中,负责识别、结合和转运气味和信息素分子到达嗅觉受体.近些年,随着各种生物新技术的应用,大量昆虫嗅觉结合蛋白被鉴定出来,其各种不同功能得到揭示.本文对近年来嗅觉结合蛋白的分子特征、蛋白结构、功能和应用等方面的研究进展进行总结和综述.总的来说,嗅觉结合蛋白...     

Unique Features of Odorant-Binding Proteins of the Parasitoid Wasp Nasonia vitripennis Revealed by Genome Annotation and Comparative Analyses

Insects are the most diverse group of animals on the planet, comprising over 90% of all metazoan life forms, and have adapted to a wide diversity of ecosystems in nearly all environments. They have evolved highly sensitive chemical senses that are central to their interaction with their environment and to communication between individuals. Understanding the molecular bases of insect olfaction is therefore of great importance from both a basic and applied perspective. Odorant binding proteins (OBPs) are some of most abundant proteins found in insect olfactory organs, where they are the first component of the olfactory transduction cascade, carrying odorant molecules to the olfactory receptors. We carried out a search for OBPs in the genome of the parasitoid wasp Nasonia vitripennis and identified 90 sequences encoding putative OBPs. This is the largest OBP family so far reported in insects. We report unique features of the N. vitripennis OBPs, including the presence and evolutionary origin of a new subfamily of double-domain OBPs (consisting of two concatenated OBP domains), the loss of conserved cysteine residues and the expression of pseudogenes. This study also demonstrates the extremely dynamic evolution of the insect OBP family: (i) the number of different OBPs can vary greatly between species; (ii) the sequences are highly diverse, sometimes as a result of positive selection pressure with even the canonical cysteines being lost; (iii) new lineage specific domain arrangements can arise, such as the double domain OBP subfamily of wasps and mosquitoes.     

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth,Amyelois transitella

Background

The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology

By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion

We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.     

绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性

气味结合蛋白(odorant binding proteins, OBPs) 在昆虫嗅觉识别中起着重要的作用, 尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors, ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制, 同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum (Meyer-Dür)气味结合蛋白AlucOBP7基因（GenBank登录号: JQ675724）, 并进行了原核表达, 以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及 6种性信息素类似物的结合能力。结果表明： 在10种棉花挥发物中, AlucOBP7能够和2 己酮及水杨酸甲酯有效结合, 结合常数分别为55.13 μmol/L和28.26 μmol/L。在6种盲蝽性信息素类似物中, 4-氧代-反-2-己烯醛和AlucOBP7 具有较强的结合能力, 结合常数为23.14 μmol/L。丁酸乙酯、 丁酸丁酯及己酸己酯也能够和AlucOBP7 有效结合, 但结合能力中等, 结合常数分别为30.58, 39.26和35.81 μmol/L。初步推测, AlucOBP7 可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins, PBPs), 并在感受性信息素和植物挥发物的过程中发挥双重功能。     

昆虫气味受体研究进展

嗅觉在昆虫的多种行为中发挥关键作用。气味分子与嗅觉神经元树突上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫的嗅觉神经元表达两类气味受体： 一是传统气味受体,该类受体同源性较低,在少部分嗅觉神经元中表达; 二是Or83b家族受体,该类受体不感受气味,在不同昆虫间较为保守且在大多数嗅觉神经元中表达。目前,对于单个传统气味受体的气味分子配体特异性所知甚少; 对于Or83b家族受体,一般认为其可能具有将传统气味受体运送至嗅觉神经元树突膜上的功能。此外,有一些实验证据不支持昆虫气味受体为G蛋白偶联受体的观点。     

Conserved odorant-binding proteins from aphids and eavesdropping predators

Background

The sesquiterpene (E)-ß-farnesene is the main component of the alarm pheromone system of various aphid species studied to date, including the English grain aphid, Sitobion avenae. Aphid natural enemies, such as the marmalade hoverfly Episyrphus balteatus and the multicolored Asian lady beetle Harmonia axyridis, eavesdrop on aphid chemical communication and utilize (E)-ß-farnesene as a kairomone to localize their immediate or offspring preys. These aphid-predator systems are important models to study how the olfactory systems of distant insect taxa process the same chemical signal. We postulated that odorant-binding proteins (OBPs), which are highly expressed in insect olfactory tissues and involved in the first step of odorant reception, have conserved regions involved in binding (E)-ß-farnesene.

Methodology

We cloned OBP genes from the English grain aphid and two major predators of this aphid species. We then expressed these proteins and compare their binding affinities to the alarm pheromone/kairomone. By using a fluorescence reporter, we tested binding of (E)-ß-farnesene and other electrophysiologically and behaviorally active compounds, including a green leaf volatile attractant.

Conclusion

We found that OBPs from disparate taxa of aphids and their predators are highly conserved proteins, with apparently no orthologue genes in other insect species. Properly folded, recombinant proteins from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-ß-farnesene with apparent high affinity. For the first time we have demonstrated that insect species belonging to distinct Orders have conserved OBPs, which specifically bind a common semiochemical and has no binding affinity for related compounds.     

昆虫化学感受蛋白(CSPs)的功能研究概述

昆虫的嗅觉系统与其各项生命活动息息相关,化学感受蛋白(CSPs)是嗅觉系统中的重要组成部分,可以结合气味或信息素分子,并传递给嗅觉受体,完成嗅觉相关功能。随着分子生物学技术和测序手段的不断发展,越来越多的昆虫CSPs得到鉴定。CSPs在昆虫体内广泛分布于触角、跗节、下颚须等化学感受器官,同时也在表皮、腹部、体躯等非感受器官大量表达,具有感知化学分子的功能并且与昆虫生长、发育、繁殖等生理功能及昆虫对杀虫剂的抗性相关。本文通过从CSPs的发现和命名、分子特性、结构及分布等方面展开综述,着重介绍CSPs的气味分子识别作用机制、抗药性机制及功能分类,以期为今后利用CSPs作为靶标防治害虫提供参考。