Immacolata Iovinella

Soluble proteins of chemical communication: an overview across arthropods

Detection of chemical signals both in insects and in vertebrates is mediated by soluble proteins, highly concentrated in olfactory organs, which bind semiochemicals and activate, with still largely unknown mechanisms, specific chemoreceptors. The same proteins are often found in structures where pheromones are synthesized and released, where they likely perform a second role in solubilizing and delivering chemical messengers in the environment. A single class of soluble polypeptides, called Odorant-Binding Proteins (OBPs) is known in vertebrates, while two have been identified in insects, OBPs and CSPs (Chemosensory Proteins). Despite their common name, OBPs of vertebrates bear no structural similarity with those of insects. We observed that in arthropods OBPs are strictly limited to insects, while a few members of the CSP family have been found in crustacean and other arthropods, where however, based on their very limited numbers, a function in chemical communication seems unlikely. The question we address in this review is whether another class of soluble proteins may have been adopted by other arthropods to perform the role of OBPs and CSPs in insects. We propose that lipid-transporter proteins of the Niemann-Pick type C2 family could represent likely candidates and report the results of an analysis of their sequences in representative species of different arthropods.

Two odorant-binding proteins mediate the behavioural response of aphids to the alarm pheromone (E)-ß-farnesene and structural analogues.

Background Aphids are agricultural pests of great economical interest. Alternatives to insecticides, using semiochemicals, are of difficult applications. In fact, sex pheromones are of little use as aphids reproduce partenogenetically most of the time. Besides, the alarm pheromone, (E)-ß-farnesene for a great number of species, is difficult to synthesize and unstable in the environment. The search for novel semiochemicals to be used in population control can be efficiently approached through the study of the olfactory system at the biochemical level. Recently odorant-binding proteins (OBPs) have been shown to play a central role in olfactory recognition, thus becoming the target of choice for designing new semiochemicals. Methodology/Principal Findings To address the question of how the alarm message is recognised at the level of OBPs, we have tested 29 compounds, including (E)-ß-farnesene, in binding assays with 6 recombinant proteins and in behaviour experiments. We have found that good repellents bind OBP3 and/or OBP7, while non repellents present different spectra of binding. These results have been verified with two species of aphids, Acyrthosiphon pisum and Myzus persicae, both using (E)-ß-farnesene as the alarm pheromone. Conclusions Our results represent further support to the idea (so far convincingly demonstrated only in Drosophila) that OBPs are involved in decoding the chemical information of odorants and pheromones, and for the first time provide such evidence in other insect species and using wild-type insects. Moreover, the data offer guidelines and protocols for the discovery of potential alarm pheromones, using ligand-binding assays as a preliminary screening before subjecting selected compounds to behaviour tests.

Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules.

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60-70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.

Structure and biotechnological applications of odorant-binding proteins

Odorant-binding proteins (OBPs) are small soluble polypeptides found in sensory organs of vertebrates and insects as well as in secretory glands and are dedicated to detection and release of chemical stimuli. OBPs of vertebrates belong to the family of lipocalin proteins, while those of insects are folded into α-helical domains. Both types of architectures are extremely stable to temperature, organic solvents and proteolytic digestion. These characteristics make OBPs suitable elements for fabricating biosensors to be used in the environment, as well as for other biotechnological applications. The affinity of OBPs for small volatile organic compounds is in the micromolar range, and they have broad specificity to a range of ligands. For biotechnological applications, OBPs can be expressed in bacterial systems at low cost and are easily purified. The large amount of information available on their structures and affinities to different molecules should allow the design of specific mutants with desired characteristics and represent a solid base for tailoring OBPs for different applications.

Diversity, abundance and sex-specific expression of chemosensory proteins in the reproductive organs of the locust Locusta migratoria manilensis.

Abstract Chemosensory proteins (CSPs) are small soluble proteins often associated with chemosensory organs in insects but include members involved in other functions, such as pheromone delivery and development. Although the CSPs of the sensory organs have been extensively studied, little is known on their functions in other parts of the body. A first screening of the available databases has identified 70 sequences encoding CSPs in the oriental locust Locusta migratoria manilensis. Applying proteomic analysis, we have identified 17 of them abundantly expressed in the female reproductive organs, but only one (CSP91) in male organs. Bacterially expressed CSP91 binds fatty acids with a specificity for oleic and linoleic acid, as well as medium-length alcohols and esters. The same acids have been detected as the main gas chromatographic peaks in the dichloromethane extracts of reproductive organs of both sexes. The abundance and the number of CSPs in female reproductive organs indicates important roles for these proteins. We cannot exclude that different functions can be associated with each of the 17 CSPs, including delivery of semiochemicals, solubilization of hormones, direct control of development, or other unknown tasks.

Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects

Odorant‐binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non‐sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol‐binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti‐inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment.

Ligand-Binding Study of Anopheles gambiae Chemosensory Proteins

Chemosensory proteins (CSPs) are a class of small proteins expressed only in arthropods and endowed with heterogeneous functions. Some of them are involved in chemical communications, others in development or other physiological roles. The numbers of CSPs in different species of insects range from 4 in Drosophila to at least 70 in locusts, whereas in other arthropods such as crustaceans and millipedes, only 2-3 very similar sequences have been reported in each species. We have expressed, in a bacterial system, 5 of the 8 CSPs predicted by the genome of the malaria mosquito Anopheles gambiae, 4 identified at the protein level (SAP1, SAP2, SAP3, and CSP3) and a fifth annotated as part of this work, obtaining the proteins with high yields and in their soluble forms. Purified CSPs have been used to study their ligand-binding properties, both using competitive binding assays and quenching of intrinsic tryptophan fluorescence, in order to get insights into their physiological functions. The agreement between the 2 sets of data supports the assumptions that the ligands, including the fluorescent reporter, bind within the core of the proteins. Their different affinities toward a set of pure chemicals suggest specific roles in chemical communication.

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.

Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria.

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.

A search for protein biomarkers links olfactory signal transduction to social immunity

BackgroundThe Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees.ResultsAfter confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect.ConclusionsOur data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.