Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier.
Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.
Briand
Loïc
L
Biochimie et Structure des Protéines, Unite de recherches INRA 477, Jouy-en-Josas Cedex, France.
Swasdipan
Nicharat
N
Nespoulous
Claude
C
Bézirard
Valérie
V
Blon
Florence
F
Huet
Jean-Claude
JC
Ebert
Paul
P
Penollet
Jean-Claude
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001516:Bees / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D018076:DNA, Complementary; D004220:Disulfides / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D019476:Insect Proteins / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D010675:Pheromones / Q000378:metabolism*; D016415:Sequence Alignment; D013229:Stearic Acids; D014364:Tryptophan / Q000378:metabolism
D018076:DNA, Complementary; D004220:Disulfides; D005456:Fluorescent Dyes; D019476:Insect Proteins; D008024:Ligands; D010675:Pheromones; D013229:Stearic Acids; C026653:12-(9-anthroyloxy)stearic acid; D014364:Tryptophan