Cloning and Expression of a Queen Pheromone-Binding Protein in the Honeybee: an Olfactory-Specific, Developmentally Regulated Protein

Abstract

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought 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 addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at ϳ2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

Olfaction in the Gypsy Moth, Lymantria dispar

“…Between 2 and 3 M PBP there is a sharp increase in pheromone binding affinity (12), which possibly reflects a micromolar dissociation constant for the PBP dimer. In several studies, dimeric PBP was observed directly in native electrophoretic gels (12,40) or by gel filtration chromatography (39,41,42). Interestingly, the monomer-dimer equilibrium shifts toward the monomer below pH 5.5 (40).…”

Transcriptomics and neuroanatomy of the clonal raider ant implicate an expanded clade of odorant receptors in chemical communication

“…Although a few studies have sought to implicate various small soluble proteins in pheromone recognition (17,(20)(21)(22)(23), it seems more likely that these proteins are involved in generic odorant solubilization and play little role in odor coding (24,25). The only known hymenopteran pheromone ligand-receptor pair to date is the honey bee (Apis mellifera) queen pheromone 9-ODA and the OR AmelOR11 (26).…”

Genome-Wide Analysis of the Odorant-Binding Protein Gene Family in Drosophila melanogaster

“…3). The ABPX subfamily also includes three beetle OBPs, the queen pheromone-binding protein from the honey bee Apis mellifera, and an OBP from the "true bug" Lygus lineolaris (Wojtasek et al 1998(Wojtasek et al , 1999Danty et al 1999;Vogt et al 1999). We have named another OBP subfamily the CRLBP family (Fig.…”

“…The five CRLBP OBPs share an average of 22% identity. The CRLBP subfamily is polyphyletic and includes two Drosophila Obps (Obp19d and Obp28a) along with OBPs from the beetle Phyllopertha diversa (P. div OBP2) and the honey bee A. mellifera (A. mel ASP2; Danty et al 1999;Wojtasek et al 1999). The sand fly Lutzomyia longipalpis salivary protein SL1 (Charlab et al 1999) clusters with a group of Drosophila OBPs, including most of those encoded by the 14 Obp genes in cytological region 56E-57A.…”

“…The ABPX subfamily includes three Drosophila OBPs (Obp83a, Obp83b, and Obp69a) as well as antennal binding proteins termed ABPXs from a variety of Lepidoptera species (Krieger et al 1996(Krieger et al , 1997, and related antennal proteins from multiple species of beetle (Wojtasek et al 1998(Wojtasek et al , 1999, the honey bee A. mellifera (Danty et al 1999), and the Hemiptera ("true bug") L. lineolaris (Vogt et al 1999). Insect OBP progenitors were likely present in ancient Neoptera, and the ABPX subfamily evidently diverged from other insect OBPs before the Endopterygota-Paraneoptera split.…”