Zhang Ld

Chemosensory proteins of Locusta migratoria

Two different classes of chemosensory proteins (CSPs) in Locusta migratoria have been identified on the basis of the molecular cloning of a series of different cDNAs from the antennae of this insect. Several CSP isoforms have been purified and biochemically characterized from antennal and wing extracts, some of them corresponding to expression products predicted for the identified cDNAs. In wings, the nature of the main endogenous ligand binding to these proteins was determined as oleoamide by a gas chromatography–mass spectrometric approach. One of these isoforms has been expressed in a bacterial system with high yield and used in a fluorescent binding assay. Competitive binding experiments have indicated the presence of long‐chain compounds among the best ligands.

Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria

Abstract. Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100°C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.

Binding properties of a locust's chemosensory protein

The chemosensory protein CSP-sg4 of the desert locust Schistocerca gregaria binds reversibly N-phenyl-1-naphthylamine in fluorescent-binding assays, with a dissociation constant of 4 microM. Upon binding to the protein, the emission peaks of the fluorescent probe undergo a marked blue shift, accompanied by an order of magnitude increase of the maximum intensity. The assay has also allowed the measurement of the affinity of CSP to other aromatic and aliphatic compounds. The binding capacity of this protein is unaffected by thermal treatments up to 100 degrees C for 20 min. The ligand-binding characteristics of chemosensory proteins may help in clarifying the role of this recently discovered class of soluble proteins in chemoreception.

Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts

Abstract.We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14xa0kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35xa0kDa in the extract of 1st–5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.

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.

Identification of the proteome composition occurring during the course of embryonic development of bees (Apis mellifera)

To investigate the proteome during embryonic development of honeybees, Apis mellifera, proteins were identified by two‐dimensional gel electrophoresis, mass spectrometry and protein engine identification tools that were applied to MASCOT and Xproteo search engines. 312, 320, 315 proteins were detected in 24, 48 and 72 h embryos. Thirty‐eight highly abundant proteins were identified at the three time points by MS fingerprinting. All 21 proteins could be identified as products of annotated genes of the honeybee. Identified proteins included six proteins related to the metabolism of carbohydrates and energy production, six proteins belonging to the heat shock protein family, three cytoskeletal proteins, four proteins related to the antioxidant system of the embryo and two proteins related to growth regulation of the embryo. Quantitative proteomics was applied to analyze differences in amounts of these proteins during the three above mentioned developmental stages. Our data present an initial molecular picture of honeybee embryos, and will hopefully pave the way for future research on this animal.

Intriguing similarities between two novel odorant-binding proteins of locusts.

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.

Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

Crystal structure of the Locusta migratoria odorant binding protein

Locusta migratoria (Lmig) causes enormous losses to agricultural products, especially because it often infests the world with great swarms as locust plagues. Locusts find their plant hosts on which they feed through their olfactory system, in which odorant binding proteins (OBPs) play an important role. Previous study indicated that the amino acid sequences of LmigOBP showed low similarity to OBPs from other insect orders and we speculated that it might perform unique binding behavior. Here, we solved the first LmigOBP1 structure at 1.65Å, which is a monomer in solution and disulfide bonds play a key role in maintaining its function. We show that LmigOBP1 possesses a unique seventh α-helix, which is located at the surface with strong interactions with the LmigOBP1 scaffold consisting of other six α-helices. Moreover, the seventh α-helix forms a wall of an L shaped internal hydrophobic cavity to accommodate linear ligands, which is consistent with the binding experiments. We also demonstrate that the ligand-binding pocket in LmigOBP1 is greatly different from that in the closest homologs mosquito OBPs. Taken together, this study provides a structural basis for designing small inhibitors to control locust.

Novel odorant-binding proteins and their expression patterns in grasshopper, Oedaleus asiaticus.

Insects use olfaction to detect exogenous odors and adapt to environments. In their olfaction systems, odorant-binding proteins (OBPs) are believed to be a key component. The unique OBP system of each species reflects the evolution of chemosensation of insects with habits. Here, we for the first time identified 15 OBPs, OasiOBP1-15, of a grasshopper, Oedaleus asiaticus, that lives in the grasslands of Northern China and is closely related to the locust, Locusta migratoria. OasiOBP9 and OasiOBP10 are specifically expressed in the antennae. Other OBPs are expressed in the antennae as well as other chemosensory organs, such as the mouthparts and wings. Significantly more OasiOBP7 was detected in male than female antennae, but there are 9 OBPs that were more expressed in female than male antennae by quantitative real-time PCR. Phylogenetic analysis indicated that most of the O.xa0asiaticus OBPs are similar to those of L.xa0migratoria, but some are substantially different. This indicates that the OBPs originally evolved in a common ancestor, but their unique chemosensory systems are adapted to different ecosystems.