Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein

Odorant-binding proteins (OBPs) are small abundant extracellular proteins t hought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in additi on to drone sex attraction. We identified, for the first time in a social i nsect, a binding protein called antennal-specific protein 1 (ASP1), which b inds at least one of the major queen pheromone components. ASP1 was charact erized by cDNA cloning, expression in Pichia pastoris, and pheromone bindin g. In situ hybridization showed that it is specifically expressed in the au xiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence re vealed it as a divergent member of the insect OBP family. The recombinant p rotein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chr omatography showed that recombinant ASP1 is dimeric. ASP1 interacts with qu een pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing elec trophoresis during development, starts at day 1 before emergence, in concom itance with the functional maturation of olfactory neurons. The isobar ASP1 b isoform appears simultaneously to ASP1a in workers, but only at similar t o 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be be cause of dimerization, which might play a physiological role in relation wi th mate attraction.