Specificity determinants of the silkworm moth sex pheromone

Abstract

The insect olfactory system, particularly the peripheral sensory system for sex pheromone reception in male moths, is highly selective, but specificity determinants at the receptor level are hitherto unknown. Using the Xenopus oocyte recording system, we conducted a thorough structure-activity relationship study with the sex pheromone receptor of the silkworm moth, Bombyx mori, BmorOR1. When co-expressed with the obligatory odorant receptor co-receptor (BmorOrco), BmorOR1 responded in a dose-dependent fashion to both bombykol and its related aldehyde, bombykal, but the threshold of the latter was about one order of magnitude higher. Solubilizing these ligands with a pheromone-binding protein (BmorPBP1) did not enhance selectivity. By contrast, both ligands were trapped by BmorPBP1 leading to dramatically reduced responses. The silkworm moth pheromone receptor was highly selective towards the stereochemistry of the conjugated diene, with robust response to the natural (10E,12Z)-isomer and very little or no response to the other three isomers. Shifting the conjugated diene towards the functional group or elongating the carbon chain rendered these molecules completely inactive. In contrast, an analogue shortened by two omega carbons elicited the same or slightly higher responses than bombykol. Flexibility of the saturated C1-C9 moiety is important for function as addition of a double or triple bond in position 4 led to reduced responses. The ligand is hypothesized to be accommodated by a large hydrophobic cavity within the helical bundle of transmembrane domains.

Figure 1. Bombykol receptor expressed in the Xenopus oocyte recording system.

Robust currents from BmorOR1•BmorOrco-expressing oocytes when perfused with bombykol, and dose-dependent responses. n = 3–5, error bars in all figures represent SEM.

Figure 2. Activation by bombykal.

(A) Current responses and (B) dose-dependent relationships obtained by challenging BmorOR1•BmorOrco-expressing oocytes with increasing concentrations of bombykol and bombykal. n = 6.

Figure 3. Chemical analysis of synthetic pheromone components.

GC-MS traces obtained from bombykol (upper trace) and bombykal (lower trace) samples freshly prepared to challenge BmorOR1•BmorOrco-expressing oocytes. Arrow indicates trace amounts of bombykal (1.3%) in the bombykol sample, whereas a dotted arrow shows traces of bombykol (0.9%) in bombykal sample. The ratio of bombykol (retention time, 16.06 min) to bombykal (15.42 min) in the two samples was 1.015±0.02, n = 3.

Figure 4. Synthetic pheromone components trapped by PBP.

(A) Traces and (B) quantification of current responses obtained from the BmorOR1•BmorOrco-expressing oocytes when presented with bombykol and bombykal solubilized either by DMSO or BmorPBP1. n = 3. *Significantly different (t-test, P<0.05).

Figure 5. Chemical structures.

Structures of the silkworm moth sex pheromone (1) and bombykol-related compounds, which were used to challenge BmorOR1•BmorOrco-expressing oocytes.

Figure 6. Stereochemical selectivity.

(A) Traces and (B) quantification of current responses from BmorOR1•BmorOrco-expressing oocytes perfused with four isomers of bombykol at 0.1 µM. n = 5. Bars with the same letter arwe not significantly different (One-way ANOVA, P<0.01).

Figure 7. Effect of altering unsaturation on receptor response.

(A) Traces and (B) quantification of current responses elicited by (8E,10Z)-hexadecadien-1-ol (9) and 10,12-hexadecadiyn-1-ol (6) presented at 1 mM. n = 3. Bars with the same letter are not significantly different (One-way ANOVA, P<0.01).

Figure 8. Effect of number of carbons distal to unsaturation.

(A) Current responses obtained by challenging BmorOR1•BmorOrco-expressing oocytes with bombykol (lower trace, positive control), (10E,12Z)-octadecadien-1-ol (7), and (10E,12Z)-tetradecadien-1-ol (8), robust response at 10 µM. (B) Dose-dependent relationships; n = 4.

Figure 9. Reducing responses by adding rigidity to the C1–C9 moiety.

(A) Current responses elicited by (10E,12Z)-hexadecadien-4-yn-1-ol (10), (4Z,10E,12Z)-hexadecatrien-1-ol (11), and bombykol (1) from BmorOR1•BmorOrco-expressing oocytes (ligands presented at 10 µM). EC₅₀s 1.7×10⁻⁵M, 1.3×10⁻⁵M, and 5.9×10⁻⁶M, respectively. (B) Dose-dependent relationships, n = 3–4.

Figure 10. Schemes A–C.

Synthetic sequence for preparation of analogues 7–11 containing the (E,Z)-dienyl moiety.

Figure 11. Schemes D–G.

Synthetic sequence for preparation of analogues 3–6 differing in unsaturation.