Xiaoli He

Characterisation of Bombyx Mori Odorant-Binding Proteins Reveals that a General Odorant-Binding Protein Discriminates between Sex Pheromone Components.

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

Genome annotation and comparative analyses of the odorant-binding proteins and chemosensory proteins in the pea aphid Acyrthosiphon pisum

Odorant‐binding proteins (OBPs) and chemosensory proteins (CSPs) are two families of small water‐soluble proteins, abundant in the aqueous fluid surrounding olfactory receptor neurons in insect antennae. OBPs are involved in the first step of olfactory signal transduction, carrying airborne semiochemicals to the odorant receptors and can be classified into three groups: Classic OBPs, Plus‐C OBPs and Atypical OBPs. Here, we identified and annotated genes encoding putative OBPs and CSPs in the pea aphid Acyrthosiphon pisum using bioinformatics. This identified genes encoding 13 Classic and two Plus‐C OBPs and 13 CSPs. Homologous OBP sequences were also identified in nine other aphid species, allowing us to compare OBPs across several aphid and non‐aphid species. We show that, although OBP sequences are divergent within a species and between different orders, there is a high similarity between orthologs within a range of aphid species. Furthermore, the phylogenetic relationships between OBP orthologs reflect the divergence of aphid evolution lineages. Our results support the ‘birth‐and‐death’ model as the major mechanism explaining aphid OBP sequence evolution, with the main force acting on the evolution being purifying selection.

Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti : genome annotation and comparative analyses

The yellow fever mosquito Aedes aegypti is an important human health pest which vectors yellow fever and dengue viruses. Olfaction plays a crucial role in its attraction to hosts and although the molecular basis of this is not well understood it is likely that odorant‐binding proteins (OBPs) are involved in the first step of molecular recognition. Based on the OBPs of Drosophila melanogaster and Anopheles gambiae we have defined sequence motifs based on OBP conserved cysteine and developed an algorithm which has allowed us to identify 66 genes encoding putative OBPs from the genome sequence and expressed sequence tags (ESTs) of Ae. aegypti. We have also identified 11 new OBP genes for An. gambiae. We have examined all of the corresponding peptide sequences for the properties of OBPs. The predicted molecular weights fall within the expected range but the predicted isoeletric points are spread over a wider range than found previously. Comparative analyses of the 66 OBP sequences of Ae. aegypti with other dipteran species reveal some mosquito‐specific genes as well as conserved homologues. The genomic organisation of Ae. aegypti OBPs suggests that a rapid expansion of OBPs has occurred, probably by gene duplication. The analyses of OBP‐containing regions for microsynteny indicate a very high synteny between Ae. aegypti and An. gambiae.

Discrimination of alarm pheromone (E)-β-farnesene by aphid odorant-binding proteins.

OBPs have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-beta-farnesene, the alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone.

Binding of the General Odorant Binding Protein of Bombyx mori BmorGOBP2 to the Moth Sex Pheromone Components

Insects use olfactory cues to locate hosts and mates. Pheromones and other semiochemicals are transported in the insect antenna by odorant-binding proteins (OBPs), which ferry the signals across the sensillum lymph to the olfactory receptors (ORs). In the silkworm, Bombyx mori (L.), two OBP subfamilies, the pheromone-binding proteins (PBPs) and the general odorant-binding proteins (GOBPs), are thought to be involved in both sensing and transporting the sex pheromone, bombykol [(10E,12Z)-hexadecadien-1-ol], and host volatiles, respectively. Quantitative examination of transcript levels showed that BmorPBP1 and BmorGOBP2 are expressed specifically at very high levels in the antennae, consistent with their involvement in olfaction. A partitioning binding assay, along with other established assays, showed that both BmorPBP1 and BmorGOBP2 bind to the main sex pheromone component, bombykol. BmorPBP1 also binds equally well to the other major pheromone component, bombykal [(10E,12Z)-hexadecadienal], whereas BmorGOBP2 discriminates between the two ligands. The pheromone analogs (10E,12Z)-hexadecadienyl acetate and (10E,12Z)-octadecadien-1-ol bind to both OBPs more strongly than does bombykol, suggesting that they could act as potential blockers of the response to sex pheromone by the male. These results are supported by further comparative studies of molecular docking, crystallographic structures, and EAG recording as a measure of biological response.

Expression of chemosensory proteins in the tsetse fly Glossina morsitans morsitans is related to female host-seeking behaviour

Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real‐time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10‐week‐old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.

Cooperative interactions between odorant-binding proteins of Anopheles gambiae

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

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.

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.