Pheromone binding to general odorant-binding proteins from the navel orangeworm

Abstract

General odorant-binding proteins (GOBPs) of moths are postulated to be involved in the reception of semiochemicals other than sex pheromones, the so-called "general odorants." We have expressed two GOBPs, AtraGOBP1 and AtraGOBP2, which were previously isolated from the antennae of the navel orangeworm, Amyelois transitella. Surprisingly, these two proteins did not bind compounds that are known to attract adult moths, particularly females. The proper folding and functionality of the recombinant proteins was inferred from circular dichroism analysis and demonstration that both GOBPs bound nonanal in a pH-dependent manner. EAG experiments demonstrated that female attractants (1-phenylethanol, propionic acid phenyl ester, and isobutyric acid phenyl ester) are detected with high sensitivity by the antennae of day-0 to day-4 adult females, with response declining in older moths. The same age-dependence was shown for male antennae responding to constituents of the sex pheromone. Interestingly, AtraGOBP2 bound the major constituent of the sex pheromone, Z11Z13-16Ald, with affinity comparable to that shown by a pheromone-binding protein, AtraPBP1. The related alcohol bound to AtraPBP1 with higher affinity than to AtraGOBP2. AtraGOBP1 bound both ligands with low but nearly the same affinity.

Fig. 1

Far-UV-CD spectra of two general-odorant binding proteins from the navel orangeworm, Amyelois transitella. AtraGOBP1, green trace; AtraGOBP2, blue trace. Note the lowest minima for AtraGOBP2 and AtraGOBP1 appeared at 209 nm and 223 nm, respectively

Fig. 2

Binding of nonanal to Amyelois transitella AtraGOBPs. This aldehyde showed high affinity to AtraGOBP1 (a) and AtraGOBP2 (b) at high pH, but no affinity at low pH. No traces of the ligand were detected in the buffer at pH 7. Histogram bars represent mean+SEM (N = 5)

Fig. 3

EAG traces recorded from antennae of live female Amyelois transitella in response to PAPE, IBAPE, 1-phenylethanol, and nonanal. All traces from top to bottom show the responses to control, and increasing doses of the test compounds (from 0.1 to 10 μg/μl source dose)

Fig. 4

Age-dependent responses of female Amyelois transitella antennae to attractants and nonanal. Responses displayed in (a), (b), and (c) were recorded with 0.1, 1, and 10 μg/μl source dose, respectively. Histogram bars represent mean+SEM (N = 10)

Fig. 5

Age-dependent responses of male Amyelois transitella antennae to the major constituent of the sex pheromone, Z11Z13-16Ald, and a key secondary constituent, Z3Z6Z9Z12Z15-23Hy. Histogram bars represent mean+SEM (N = 10)

Fig. 6

Binding of the aldehyde and alcohol constituents of the Amyelois transitella sex pheromone system to male and female olfactory proteins. a test ligand, Z11Z13-16Ald and b test ligand, Z11Z13-16OH. The high affinity of the aldehyde to AtraPBP1 and AtraGOBP2 is comparable (a), whereas the alcohol showed higher affinity for AtraPBP1 than AtraGOBP2. AtraGOBP1 bound both aldehyde and alcohol with low but comparable affinity. Histogram bars represent mean+SEM (N = 5)