Edited by: Catherine Loudon, University of California, Irvine, USA
Reviewed by: Steven Sinkins, Lancaster University, UK; Jesper Givskov Sørensen, Aarhus University, Denmark
*Correspondence: Walter S. Leal, Department of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA e-mail:
This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology.
†Present address: Jiao Yin, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China;
Hongxia Duan, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China
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As opposed to humans, insects rely heavily on an acute olfactory system for survival and reproduction. Two major types of olfactory proteins, namely, odorant-binding proteins (OBPs) and odorant receptors (ORs), may contribute to the selectivity and sensitivity of the insects' olfactory system. Here, we aimed at addressing the question whether OBPs highly enriched in the antennae of the southern house mosquito,
In insects, olfaction is essential for survival and reproduction (Leal,
Diseases transmitted by mosquitoes destroy more lives on a year basis than war, terrorism, gun violence, and other human maladies combined (Leal,
Selectivity of OBPs is typically investigated by comparing their binding affinities for physiologically relevant compounds, i.e., by inferring their ability to carry preferentially certain ligands than others. For example, binding affinity of an OBP from the malaria mosquito
Our biochemical, biophysical and structural studies suggest that a pH-mediated conformational change leads to the delivery of odorant to receptors, although there is another school that support receptor activation by OBP-odorant complexes (Leal,
This research was designed to investigate the potential role of OBPs on the selectivity of the southern house mosquito olfactory system by a three-prong approach. First, we selected 5 OBPs with the highest differential transcript levels in antennae (vs. legs) to clone, express, and test their binding affinity against a panel of 34 physiologically relevant compounds. Then, we studied the effect of pH on the binding of the mosquito oviposition pheromone to CquiOBP1. Lastly, we docked into CquiOBP1 the ligands with the highest affinities to study their interactions in the binding site.
Mosquitoes were raised, as previously described (Xu et al.,
CquiOBP2-Fw (
CquiOBP2- Rv (
CquiOBP3-Fw (
CquiOBP3- Rv (
CquiOBP5-Fw (
CquiOBP5-Rv (
CquiOBP11-Fw (
CquiOBP11-Rv (
PCR reactions were carried out using Advantage GC 2 PCR Kit (Clontech, Mountain View, CA). PCR products were purified by QIAquick gel extraction kit (Qiagen, Valencia, CA) and then cloned into pGEM-T vector (Promega, Madison, WI). After screening colonies, plasmids were extracted using the QIAprep Spin Miniprep kit (Qiagen) and sequenced by ABI 3730 automated DNA sequencer at Davis Sequencing (Davis, CA).
One microgram of pET-22b(+) vector (EMD Chemicals,Gibbstown, NJ) was digested with 6 U of
CquiOBP1 was expressed following our previous protocol (Leal et al.,
Binding affinity (Ban et al.,
Fluorescence spectra were recorded in a right angle configuration on a spectrofluorimeter (RF-5301, Shimadzu, Kyoto, Japan) at room temperature using a 1 cm light path fluorimeter quartz cuvette. Slit widths of 10 nm were selected for both excitation and emission. N-phenyl-1-naphthylamine (NPN, or 1-NPN) (Ban et al.,
To investigate the effects of pH on CquiOBP1–ligand complexes, fluorescence binding assay was measured between CquiOBP1 and three best ligands at different pH values. Buffer were prepared into two different ways: starting from 1M phosphate buffer, pH 4, pH was increased and adjusted by adding sodium hydroxide. Then protein was added to make a solution at each tested pH from 4.5 to 8. Experiments were repeated starting from pH 8 and lowering the pH by adding hydrochloric acid. Each tested solution had 200 mM buffer and 10 μg/ml (microgram) of CquiOBP1. As there were no significant differences in fluorescence data generated at a certain pH regardless of having the pH raised by adding NaOH or lowered by addition of HCl, data were pooled for subsequent analysis.
Molecular docking was performed with Surflex-dock module in Sybyl vs.7.3 software (Tripos Associates, St. Louis, MO). To determine the suitability of our approach, we first compared the crystal structure of CquiOBP1-MOP complex (Mao et al.,
CquiOBP1 is the first olfactory protein identified from mosquitoes by conventional biochemical approach, i.e., isolation, N-terminal sequencing and subsequent cDNA cloning with degenerate primers (Ishida et al.,
All OBP cDNAs cloned were identical to those reported in GenBank, specifically CquiOBP2: FJ947084, 146 aa residues, including a signal peptide (Petersen et al.,
Expression of CquiOBP3 at different temperatures and different IPTG concentrations gave low yields. Even at the best conditions, the yield was so low that the target protein was lost during purification attempts. By contrast, CquiOBP2 and CquiOBP5 were obtained at high yields, as previously reported for CquiOBP1. While large-scale expression of CquiOBP1 and CquiOBP2 gave the highest yields at 28°C, 1–3 h after induction with IPTG, optimal expression of CquiOBP5 was achieved under the same conditions but at 37°C. Highly purified CquiOBP1, CquiOBP2, and CquiOBP5 were used for subsequent binding assays.
The three OBPs tested show affinity for NPN within the normal range reported in the literature (Ban et al.,
DEET | – | 82 | – | – | 96 | – | – | 92 | – |
PMD | – | 80 | – | 5.3 | 35 | 1.3 | 13.8 | 61 | 9.6 |
Picaridin | 10.2 | 51 | 6.4 | – | 77 | – | 7.9 | 45 | 5.6 |
IR3535 | – | 71 | – | – | 90 | – | – | 67 | – |
Skatole | – | 75 | – | 5.6 | 31 | 1.4 | 14.4 | 62 | 10.1 |
Indole | – | 86 | – | – | 87 | – | – | 80 | – |
MOP | 8.4 | 48 | 5.3 | – | 86 | – | 10.3 | 52 | 7.2 |
4-Methylphenol | – | 73 | – | – | 85 | – | – | 90 | – |
4-Ethylphenol | – | 85 | – | – | 91 | – | – | 84 | – |
( |
– | 79 | – | – | 93 | – | – | 83 | – |
1-Octen-3-ol | – | 69 | – | – | 93 | – | 11.6 | 59 | 8.1 |
3-Octanol | – | 87 | – | – | 78 | – | – | 85 | – |
1-Hexanol | – | 85 | – | – | 89 | – | – | 88 | – |
(±)-Citronellal | – | 65 | – | – | 88 | – | – | 90 | – |
Ethyl 2-phenylacetate | – | 92 | – | – | 87 | – | – | 91 | – |
Sulcatone | – | 87 | – | – | 84 | – | – | 66 | – |
4-Methylcyclohexanol | – | 88 | – | – | 88 | – | – | 93 | – |
Methyl salicylate | – | 87 | – | – | 72 | – | – | 89 | – |
Geranyl acetate | – | 94 | – | – | 90 | – | – | 81 | – |
Eugenol | – | 72 | – | – | 82 | – | 10.7 | 57.5 | 7.4 |
Cyclohexanone | – | 79 | – | – | 86 | – | – | 82 | – |
Acetophenone | – | 88 | – | – | 92 | – | – | 87 | – |
Thujone | – | 96 | – | – | 87 | – | – | 90 | – |
Benzaldehyde | – | 83 | – | – | 91 | – | – | 93 | – |
3,5-Dimethylphenol | – | 84 | – | 7.4 | 40 | 1.9 | – | 93 | – |
1,2-Dimethoxybenzene | – | 88 | – | – | 91 | – | – | 87 | – |
2-Phenylethanol | – | 91 | – | – | 92 | – | – | 95 | – |
Linalool oxide | – | 92 | – | – | 95 | – | – | 94 | – |
Linalool | – | 91 | – | – | 97 | – | – | 86 | – |
Eucalyptol | – | 96 | – | – | 90 | – | – | 73 | – |
Nonanal | 10.5 | 51.4 | 6.6 | – | 87 | – | 8.3 | 46.5 | 5.8 |
Camphor | – | 90 | – | – | 85 | – | – | 89 | – |
1-Octyn-3-ol | – | 66 | – | – | 92 | – | 12.8 | 62.2 | 8.9 |
Fenchone | – | 89 | – | – | 87 | – | – | 92 | – |
The dissociation constants for the best ligands for CquiOBP1, namely, MOP (Laurence and Pickett,
The pH dependent curves for CquiOBP1 obtained with the three best ligands showed a nearly bell-shape curve, with three different maxima (Figure
To assess the suitability of dock simulations in providing insights into the interactions of nonanal and picaridin with CquiOBP1, we first re-docked MOP into CquiOBP1 and compared this structure with the previously reported crystal structure (Figure
Using a fluorescence reporter and a panel of 34 physiologically relevant compounds, we measured binding affinities of three major OBPs from the southern house mosquito, namely, CquiOBP1, CquiOBP2, and CquiOBP5. Based on dissociation constants, we hypothesized that CquiOBP2 is a carrier for the oviposition attractant skatole, and CquiOBP1 and CquiOBP5 might transport the oviposition pheromone MOP, a human-derived attractant nonanal, and the insect repellent picardin. Examination of binding of these three ligands to CquiOBP1 at various pH values suggests that CquiOBP1 might discriminate MOP from nonanal/picaridin on the basis of the midpoint transition of a pH-dependence conformational change. Additionally, docking studies suggest MOP is better accommodated in the binding cavity than the other two ligands. Taken together, these findings suggest that OBPs may be involved in the selectivity of the mosquito olfactory system, but this may not be manifested clearly in binding affinities.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank lab members for assistance, particularly Dr. Pingxi Xu, for his helping with the design of pH-dependent binding studies, and Dr. Anthon Cornel (UC Davis) for providing mosquitoes that allowed us to duplicate his colony at the Davis campus. This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award R01AI095514. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. JI and HD sabbatical leaves in Davis were supported in part by CSC—China Scholarship Council.