Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay

Pheromone-binding proteins (PBPs), located in the sensillum lymph of pherom one-responsive antennal hairs, are thought to transport the hydrophobic phe romones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of s pecies-specific pheromones, We have investigated the binding properties and specificity of PBPs from Mamestra brassicae (MbraPBP1), Antheraea polyphem us (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1- M6), mutated at residues of the internal cavity to mimic that of BmorPBP, u sing the fluorescence probe 1-aminoanthracene (AMA), AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1 -M6. The latter result indicates that relatively limited modifications to t he PBP cavity actually interfere with AMA binding, suggesting that ARIA bin ds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence o f specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings brin g into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds.