Sensillum-lymph proteins from antennal olfactory hairs of the moth Antheraea polyphemus (Saturniidae)

Klein, Uwe

doi:10.1016/0020-1790(87)90093-x

Cited by 145 publications

(97 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Soluble protein could be purified readily in two steps by preparative IEF followed by gel filtration; insoluble protein could be solubilized, refolded, and purified by the same two steps. The analysis of the purified, soluble rPBP, as well as the refolded rPBP, indicates that they have the molecular size, immunoreactivity, and isoelectric point identical to that of native A. polyphemus PBP (Klein, 1987). Most importantly from a physiological point of view, both soluble and refolded rPBP have identical binding affinities for pheromone as determined by photoaffinity labeling experiments.…”

Section: Discussionmentioning

confidence: 86%

“…Thus, 55 mL of desalted soluble protein containing 2% Pharmalyte 4-6 (or Biolyte 4/6) ampholytes was focused into 20 chambers at 12 W constant power, 300-1, OOO V, 4 "C, during 6 h. The fractions were harvested by suction and analyzed in three ways: pH, immunoreactivity, and SDS-PAGE. The pH 3.8-5.0 range was expected to contain Apo-3 protein, whose predicted PI value is 4.43 (Raming et al, 1989) and measured PI is 4.7 (Klein, 1987). Dot blots on PVDF were performed for each fraction, for crude protein, and with known amounts of native purified Apo-3 protein.…”

Section: Cell Culture Induction and Protein Purificationmentioning

confidence: 99%

See 1 more Smart Citation

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Prestwich

1993

Protein Science

View full text Add to dashboard Cite

The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl 6-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave >95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E, 11Zhexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure.Keywords: Antheraea polyphemus; expression cassette PCR; Lepidoptera; olfactory signal transduction; pheromone binding protein; pheromone perception; photoaffinity labeling; protein refoldingInsects offer an ideal model system for studying the molecular details of olfaction. Mate-seeking behavior in moths (Insecta, Lepidoptera) is under strong selection pressure, and each moth species responds with exquisite sensitivity to a limited number of fatty acid-derived pheromones. Perception of the female sex pheromone by male moths is mediated by proteins located in the sensory hairs of the antennae (Vogt, 1987;Vogt et al., 1990b;Prestwich, 1992). These include soluble pheromone binding

show abstract

Section: Discussionmentioning

confidence: 86%

Section: Cell Culture Induction and Protein Purificationmentioning

confidence: 99%

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Prestwich

1993

Protein Science

View full text Add to dashboard Cite

show abstract

“…In moths, PBPs are expressed at high concentrations in the pheromone-detecting sensilla. It has been estimated that in the wild silkmoth, Antheraea polyphemus, the concentration of a PBP is as high as 10 mM (20,21). By comparing the amounts of protein extracted from male antennae of the silkworm moth with pure recombinant BmorPBP, we estimated that in the B. mori pheromone-detecting sensilla, BmorPBP is expressed at Ϸ3 mM (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Pheromone reception in fruit flies expressing a moth's odorant receptor

Syed

Ishida

Taylor

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

We have expressed a male-specific, pheromone-sensitive odorant receptor (OR), BmorOR1, from the silkworm moth Bombyx mori in an ''empty neuron'' housed in the ab3 sensilla of a Drosophila ⌬halo mutant. Single-sensillum recordings showed that the BmorOR1-expressing neurons in the transgenic flies responded to the B. mori pheromone bombykol, albeit with low sensitivity. These transgenic flies responded to lower doses of bombykol in an altered stimulation method with direct delivery of pheromone into the sensillum milieu. We also expressed a B. mori pheromonebinding protein, BmorPBP, in the BmorOR1-expressing ab3 sensilla. Despite the low levels of BmorPBP expression, flies carrying both BmorOR1 and BmorPBP showed significantly higher electrophysiological responses than BmorOR1 flies. Both types of BmorOR1-expressing flies responded to bombykol, and to a lesser extent to a second compound, bombykal, even without the addition of organic solvents to the recording electrode buffer. When the semiochemicals were delivered by the conventional puffing of stimulus on the antennae, the receptor responded to bombykol but not to bombykal. The onset of response was remarkably slow, and neural activity extended for an unusually long time (>1 min) after the end of stimulus delivery. We hypothesize that BmorOR1-expressing ab3 sensilla lack a pheromone-degrading enzyme to rapidly inactivate bombykol and terminate the signal. We also found an endogenous receptor in one of the sensillum types on Drosophila antenna that responds to bombykol and bombykal with sensitivity comparable to the pheromone-detecting sensilla on B. mori male antennae.BmorOR1 ͉ BmorPBP ͉ olfaction ͉ signal termination ͉ single-sensillum recordings T he exquisite olfactory system of insects has been intriguing to scientists, particularly since the observation early in the last century that male peacock moths were attracted to female moths and probably flew from several kilometers away to find mates (1). With the discovery of the first sex pheromone from the silkworm moth, Bombyx mori (2), it became evident that insects rely on semiochemicals for the recognition not only of potential mates but also, for example, of prey and of specific features of the environment. An array of 17,000 sensilla (3) on the antennae of the silkworm moth detect not only the major constituent of the sex pheromone, bombykol, but also a second compound, bombykal, that is released by the female pheromone gland (4). These pheromone-detecting sensilla house two olfactory receptor neurons (ORNs), one specifically tuned to bombykol and the other to bombykal (4). The selectivity and sensitivity of the system are so remarkable that minimal structural modifications to pheromone molecules render them inactive (5), whereas a single molecule of the natural product is estimated to be sufficient to activate neurons in male antennae (6). Although odorant receptors (ORs) from the silkworm moth have been isolated (7,8), expressed in heterologous cell systems, and demonstrated to be activated by bombykol ...

show abstract

“…The ''on'' rate of bombykol binding to BmorPBP is very small when compared, for example, to the ''on'' rates for the binding of fatty acids to their binding proteins (k on Ϸ 10 M Ϫ1 ⅐s Ϫ1 ) (27). However, the small rate is compensated for by high concentrations (10 mM) of PBP in the sensillar lymph (28). Under these physiological conditions, the t 1/2 of the unbound bombykol is only Ϸ1 ms. A fast uptake of pheromone is important for the fast rise of the receptor potential and a fast onset of the nerve impulse (11).…”

Section: Discussionmentioning

confidence: 99%

Kinetics and molecular properties of pheromone binding and release

Leal

Chen

Ishida

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

167

139

View full text Add to dashboard Cite

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an ''on'' rate of 0.068 ؎ 0.01 M ؊1 ⅐s ؊1 . With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is Ϸ1 ms. A pH-dependent conformational change (BmorPBP B 3 BmorPBP A ), associated with the release of pheromone, is a first-order reaction (k ‫؍‬ 74.1 ؎ 0.32 s ؊1 ; t1/2, 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K D ‫؍‬ 105 nM) at pH 7 but low affinity (K D ‫؍‬ 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBP⌬P129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.Bombyx mori pheromone-binding protein ͉ bombykol ͉ effect of C terminus on pheromone release ͉ fast uptake of pheromone and delivery ͉ mutated pheromone-binding proteins F or many insects, small-molecule signals communicate the availability of food, the presence of friends and foes, and the readiness to mate. In general, mate-finding is an essential prerequisite for exploring insects' enormous reproductive potential and, consequently, leads to their domination of the terrestrial world. To advertise their readiness to mate, female moths, for example, produce and release sex pheromones. Only minute amounts are released, so as to avoid chemical conspicuousness. Once released, the chemical signals are diluted in the environment and mixed with a myriad of physiologically irrelevant compounds, including pheromones from other species. Even though each species communicates with a specific pheromonic language, males can detect the low-level signals from conspecific females because of a highly developed olfactory system. To find females and successfully reproduce, males may have to make long-distance, odorant-oriented flights. Navigation requires a highly selective, sensitive, and dynamic sensory system for the detection of pheromones. Given the structure of pheromone plumes (1), insects have only a few milliseconds to reset their detectors while flying in the clean air spaces between pockets of chemical signals.Pheromones are largely hydrophobic compounds, whereas pheromo...

show abstract

Sensillum-lymph proteins from antennal olfactory hairs of the moth Antheraea polyphemus (Saturniidae)

Cited by 145 publications

References 16 publications

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Pheromone reception in fruit flies expressing a moth's odorant receptor

Kinetics and molecular properties of pheromone binding and release

Contact Info

Product

Resources

About