Fatty acid solubilizer from the oral disk of the blowfly

Abstract

Background: Blowflies are economic pests of the wool industry and potential vectors for epidemics. The establishment of a pesticide-free, environmentally friendly blowfly control strategy is necessary. Blowflies must feed on meat in order to initiate the cascade of events that are involved in reproduction including juvenile hormone synthesis, vitellogenesis, and mating. During feeding blowflies regurgitate salivary lipase, which may play a role in releasing fatty acids from triglycerides that are found in food. However, long-chain fatty acids show low solubility in aqueous solutions. In order to solubilize and ingest the released hydrophobic fatty acids, the blowflies must use a solubilizer.

Methodology: We applied native PAGE, Edman degradation, cDNA cloning, and RT-PCR to characterize a protein that accumulated in the oral disk of the black blowfly, Phormia regina. In situ hybridization was carried out to localize the expression at the cellular level. A fluorescence competitive binding assay was used to identify potential ligands of this protein.

Conclusion: A protein newly identified from P. regina (PregOBP56a) belonged to the classic odorant-binding protein (OBP) family. This gene was expressed in a cluster of cells that was localized between pseudotracheae on the oral disk, which are not accessory cells of the taste peg chemosensory sensilla that normally synthesize OBPs. At pH 7 and pH 6, PregOBP56a bound palmitic, stearic, oleic, and linoleic acids, that are mainly found in chicken meat. The binding affinity of PregOBP56a decreased at pH 5. We propose that PregOBP56a is a protein that solubilizes fatty acids during feeding and subsequently helps to deliver the fatty acids to the midgut where it may help in the process of reproduction. As such, PregOBP56a is a potential molecular target for controlling the blowfly.

Figure 1. Identification of an oral disk-specific protein by native PAGE analysis.

Protein was extracted from each experimental tissue of females at day 3 adult stage, separated by 15% native PAGE, and visualized with Coomassie Brilliant Blue-R250 staining. The source of the protein is shown at top of the gel profile. The number of individuals from which the tissues were collected is indicated in parenthesis. The arrow and arrowhead indicate the migration of an oral disk-specific protein and a chemical sense-related lipophilic ligand-binding protein (CRLBP), respectively.

Figure 2. Nucleotide and deduced amino acid sequences and phylogenetic analysis of oral disk-specific protein (PregOBP56a).

A, cDNA and deduced amino acid sequences of an oral disk-specific protein. A signal peptide is shown in italic. N-terminal amino acid sequence determined by Edman degradation is underlined. Arrowheads show 6 cysteine residues, which are hallmarks of insect classic OBPs. The three arrows indicate annealing sites of a degenerate (EEQKAKV-1) and two gene-specific (PregOBP56a-1 and PregOBP56a-2) primers, B, Phylogenetic tree of odorant-binding protein 56a like-protein sequences. Phormia regina odorant-binding protein 56a is shown in bold. Accession numbers of each protein are given in the parenthesis. Bootstrap values were determined from 1,000 replications. Bar indicates 5% divergence.

Figure 3. Expression of PregOBP56a.

A, Tissue specificity of PregOBP56a expression. cDNA was synthesized from total RNA extracted from each experimental tissue at day 0 adult stage. Gene expression was detected by RT-PCR. actin expression was used as an internal control. PregOBP56a was highly expressed in the oral disk. In contrast CRLBP, an OBP related to chemical reception, was expressed in the oral disk and antenna. B, Cellular localization of PregOBP56a expression in the oral disk by in situ hybridization. Sense and anti-sense probes were prepared by pSTP18 inserted with a PCR product amplified by primers PregOBP56a-1 and PregOBP56a-2. The left panel shows the localization of the oral disk within the head of a female. The right panel shows the localization of PregOBP56a transcripts in a cluster of cells that are localized between pseudotracheae. Scale bar is 20 µm.

Figure 4. Far UV circular dichroism spectra of purified recombinant PregOBP56a.

A positive peak of the CD spectra at 194 nm and two negative peaks at 209 and 221 nm are observed at pH 7 and pH 5, indicating that PregOBP56a is an α-helix-rich protein as other insect OBPs.

Figure 5. Binding of PregOBP56a to N-phenyl-1-naphthylamine (1-NPN) and potential ligands derived from chicken meat.

A, A typical emission spectra following the addition of 1-NPN (3.2–16 µM final concentration) to recombinant PregOBP56a (10 µg/ml of 20 mM phosphate-citrate buffer, pH 7). The emission spectra were generated following excitation at a wavelength of 337 nm. B, Binding of 1-NPN to PregOBP56a at pH 7, 6, and 5. In these experiments PregOBP56a (10 µg/ml) was incubated with various concentrations (1.6–19.2 µM) of 1-NPN and emission at 440 nm was plotted. Values are means ± standard deviation, n = 3. C, Typical emission spectra following the addition of a competitor to PregOBP56a bound to 1-NPN. PregOBP56a (10 µg/ml) in 20 mM phosphate-citrate buffer at pH 7 was pre-incubated with 16 µM of 1-NPN (final concentration). Then, palmitic acid (0–3.2 µM final concentration) was added to the reaction, and the emission spectra (excitation a wavelength of 337 nm) were recorded. D, Binding of PregOBP56a to various ligands at pH 7. For each set of data, fluorescence values at a wavelength of 440 nm were plotted as percent of that obtained in the absence of the competitor. Values are means ± standard deviation, n = 3. The competitors are shown as insets. PregOBP56a bound palmitic, stearic, oleic, and linoleic acids but not decanoic acid, palmityl acetate, or cholesterol. E, Binding of PregOBP56a to various ligands at pH 6. The results were almost identical to those in panel D. F, Binding of PregOBP56a to various ligands at pH 5. The binding affinity of stearic acid decreased to same level as decanoic acid, palmityl acetate, and cholesterol. Due to low binding to PregOBP56a, the IC₅₀ of other test competitors was not obtained.