Ligand binding to six recombinant pheromone-binding proteins of Antheraea polyphemus and Antheraea pernyi

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, (E,Z)-6,11-hexadecadienyl acetate (H-3-Ac1) and (E,Z)-6,11-hexadecadienal (H-3-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone (H-3-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of H-3-Ac1. In competition experiments with H-3-Ac1 and the third unlabelled pheromone component, (E,Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound H-3-Ald only. All of the six PBPs strongly bound H-3-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for H-3-Ac1 and H-3-Ald bound to PBPs. No pH influence was found for H-3-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1-3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation.