Tài liệu Báo cáo khoa học: Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier pdf

Characterization of a chemosensory protein (ASP3c) from honeybee ( Apis mellifera L.) as a brood pheromone carrier Loı¨c Briand 1 , Nicharat Swasdipan 2 , Claude Nespoulous 1 , Vale ´ rie Be ´ zirard 1 , Florence Blon 1 , Jean-Claude Huet 1 , Paul Ebert 2 and Jean-Claude Pernollet 1 1 Biochimie et Structure des Prote ´ ines, Unite ´ de recherches INRA 477, Jouy-en-Josas Cedex, France; 2 Department of Biochemistry and Molecular Biology, University of Queensland, St Lucia, Australia Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insectspecies,whicharebelievedtobeinvolvedinchemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)–Cys(II), Cys(III)–Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of a-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural phero- monal ligand bound by a recombinant CSP with a measured affinity constant. Keywords: Apis mellifera L.; brood pheromone; chemosen- sory protein; lipid-binding protein; olfaction. In insect antennae, the first step in chemical detection is the transport of hydrophobic signalling molecules by olfactory- binding proteins (OBPs) to receptor neurons through the sensillum lymph [1–3]. Insect OBPs are small acidic soluble proteins (13–16 kDa), highly concentrated in the sensillum lymph. They can be roughly classified as pheromone- binding proteins (PBPs) and general odorant-binding pro- teins. PBPs are supposed to be involved in sex pheromone detection, although recent findings have brought into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal components [4]. In contrast, general OBPs seem to play a more general role in olfaction by carrying odorant molecules [5]. Although the physiological function of OBPs is not yet well understood, their essential role in eliciting the behavioral response and odor coding have been demonstrated in the fruit fly [6–9] and in the fire ant [10]. Another class of soluble chemosensory proteins (CSPs), which share no sequence homology with either PBPs or general OBPs, has been described in insects. Such proteins have been observed in antennae of most orders of insects such as Diptera [11–13], Lepidoptera [14–19], Hymenoptera [20], Coleoptera [21], Blattoidea [22], Orthoptera [23,24] and Phasmida [25–27]. Their occurrence is generally associated with chemosensory organs, such as legs and palpi [16,19,20,23,28,29]. They also were expressed in other sites of the insect body, such as Drosophila melanogaster ejaculatory bulb [30], Mamestra brassicae proboscis [17], labial palps of the moth Cactoblastis cactorum [14] and cells underlying the cuticle in Phasmatodea and Orthoptera [31]. Although they have not yet been demonstrated to play an olfactory role, their tissue location and initial ligand binding data both support the hypothesis that CSPs are involved in chemoreception. Their natural ligands have not yet been determined, although binding data indicate that CSPs bind highly hydrophobic linear molecules similar to insect pheromones and fatty acids [31,32]. CSPs do not share any structural similarity to insect PBPs and general OBPs. They are smaller proteins (100–110 amino acid residues) containing four cysteines instead of six with conserved interval spacing involved in two disulfide bonds [23,31]. CSPs from M. brassicae and Schistocerca gregaria have been expressed in Escherichia coli and structurally charac- terized [31–34]. They are monomers with a high a-helical content, as shown by CD and NMR spectroscopy [31,34]. This was recently supported by the report of the first CSP tridimensional structure, that of the moth M. brassicae, Correspondence to J C. Pernollet, Biochimie et Structure des Prote ´ ines, Unite ´ de recherches INRA 477, Domaine de Vilvert, F-78352, Jouy-en-Josas Cedex, France. Fax: 33 1 34 65 27 65, Tel.: 33 1 34 65 27 50, E-mail: pernolle@jouy.inra.fr Abbreviations:ASA,(+/–)-12-(9-anthroyloxy)stearic acid; ASP, antennal specific protein; BrC15-Ac, 15-bromopentadecanoic acid; C14-Ac, myristic acid; C16-Ac, palmitic acid; C18-Ac, stearic acid; C16-Me, methyl palmitate; C18-Me, methyl stearate; CSP, chemo- sensory protein; OBP, odorant-binding protein; PBP, pheromone- binding protein; RPLC, reversed phase liquid chromatography. Enzyme: Trypsin (EC 3.4.21.4). Note: Nucleotide sequence of ASP3c has been deposited in the GenBank Sequence Database with accession number AF481963. (Received 1 July 2002, accepted 30 July 2002) Eur. J. Biochem. 269, 4586–4596 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03156.x which exhibits a novel type of a-helical fold with six helices connected by a–a loops [32]. The honeybee (Apis mellifera L.) is able to discriminate among a wide range of odorants [35,36]. Its OBPs, which are evolutionary divergent from the Lepidopteran OBPs [37], were classified into three subclasses of antennal- specific proteins (ASP), namely ASP1, ASP2 and ASP3 [20,38]. ASP1 has been shown to be associated with queen pheromone detection because of its higher abundance in drone, its location in sensilla placodea and ability to bind 9-keto-2(E)-decenoic acid and 9-hydroxy-2(E)-decenoic acid [38,39], the most active components of the queen pheromone blend [40,41]. Based on sequence similarity, tissue-specificity and odorant binding experiments, ASP2, which does not bind any of these queen pheromone components [39], was assigned to be a member of the insect general OBP family [42]. In contrast, the ASP3 subclass was classified as a CSP family due to N-terminal sequence homology [20]. Recently, we purified natural ASP3c, which is com- monly found in drones and workers and was observed as a soluble protein of 12 757.1 ± 0.3 Da. In the present work we report its cloning, sequencing and heterologous expression using the yeast Pichia pastoris. Several struc- tural features of recombinant ASP3c such as its disulfide bridge pattern, secondary and quaternary structures were determined. We showed using a fluorescent probe binding assay that ASP3c is able to interact with fatty acids and brood pheromone components. This report relates the first affinity constant for a pheromonal ligand bound by an insect CSP. EXPERIMENTAL PROCEDURES Strains and materials Escherichia coli strain DH5a was used for DNA subcloning and propagation of the recombinant plasmid. Pichia pastoris strain GS115 (his4) was used in the expression study. Oligonucleotides were synthesized by MGW Biotech (France). pPIC3,5K was purchased from Invitrogen (France). Origins of chemicals are indicated in the text. cDNA cloning of ASP3c Antennae were collected from 7500 adult worker bees and poly (A +) mRNA isolated using the Quick mRNA Purification Kit (Pharmacia). A cDNA library of 10 5 primary recombinants was generated from poly (A+) mRNA using the Capfinder (Clontech) cDNA cloning system and the kZAP II cloning vector (Stratagene). DNA sequencing was performed on 19 clones after in vitro excision (Stratagene) using the services of the Australian genome research facility. Sequence analysis Related protein sequences were identified using the Basic Local Alignment Search Tool (BLAST 2.0) computed at the Swiss Institute of Bioinformatics. Sequence alignment was performed with CLUSTAL W using the Blosum 50 homology matrix and per cent amino acid sequence identity was calculated [43]. Construction of the expression vector The cDNA encoding the precursor ASP3c with its native signal peptide was amplified by PCR using the following primers: 5¢ primer, 5¢-GAGCCCGGATCCACCATGAA GGTCTCAATAATT 3¢;3¢ primer, 5¢-CTGACG GAAT TCTTAAACATTAATGCC 3¢. These primers encoded a Kozak consensus sequence as well as BamHI and EcoRI restriction sites. The PCR-amplified fragment was cloned into the BamHI and EcoRI sites of pPIC3,5K and the integrity of the resulting construct was confirmed by DNA sequencing. Transformation of Pichia pastoris and screening for ASP3c expression The expression plasmid was linearized with BglII and transferred into the Pichia pastoris yeast host by the electroporation method as described in the manual (version 3.0) of the Pichia expression Kit (Invitrogen). The selection of multicopy integrants was achieved by using increased levels (0.5–2 mgÆmL )1 ) of G418 (Clontech, Ozyme, France). Large scale protein production was achieved as recently described [44] except that the protein was secreted for only 3 days using buffered minimal MeOH medium at pH 8.0 supplemented with 2% tryptone (Sigma) and 5 m M EDTA. During the induction period, MeOH was fed twice a day in order to maintain a concentration of 0.5% v/v. Purification of the recombinant ASP3c ASP3c was purified by reversed phase liquid chromatogra- phy (RPLC). After removing insoluble components from supernatant containing recombinant proteins by filtration, the solutions were dialyzed 3 days at 4 °C, using a dialysis tube with 8000 Da cut off (Servapor, Polylabo, France) and lyophilized. Purifications were performed using an Aqua- pore C8 column (Prep )10, 1.0 i.d. · 3.0 cm, Perkin Elmer, France). The lyophilized supernatant was resuspended in eluent A (25 m M ammonium acetate, pH 7.0) and the column, equilibrated with the same eluent. After loading the sample, the column was washed extensively with eluent A. Elution was then achieved using a linear gradient to 33.3% eluent B (25 m M ammonium acetate, pH 7.0, 60% v/v acetonitrile in H 2 O) in the first 15 min, to 66.6% B in the next 40 min and to 100% eluent B, in the last 10 min. The flow rate was 2.5 mLÆmin )1 and the absorbance was recorded at 280 nm. The fractions containing purified proteins were pooled, dialyzed extensively against MilliQ H 2 O and lyophilized. Recombinant ASP3c characterization SDS/PAGE (16% acrylamide) was performed using a Mini- Protean II system (Bio-Rad, France) [45]. The molecular mass calibration kits low range and polypeptides (Bio-Rad) were used and the proteins stained with Serva blue G. ASP3c was analyzed by MALDI-TOF mass spectrometry. Two microlitres of purified ASP3c were mixed with 2 lLof matrix solution (saturated solution of sinapinic acid in 30% v/v acetonitrile, 0.2% v/v trifluoroacetic acid). One micro- litre of the mixture was applied to a stainless steel sample plate and allowed to air dry. Mass calibration was made Ó FEBS 2002 Brood pheromone binding by bee chemosensory protein (Eur. J. Biochem. 269) 4587 with the calibration mixture 2 (PE Biosystems) using thioredoxin from Escherichia coli at 11 674.48 Da [M + H] + and apomyoglobin from horse at 16 952.56 Da [M + H] + . Mass spectra were obtained using a PE Biosystems Voyager-DE STR+ spectrometer in linear mode. N-terminal amino acid sequence analysis of proteins was performed by automated Edman degradation using a Perkin-Elmer Procise 494-HT protein sequencer with reagents and methods of the manufacturer. Oligomerization of the undenatured recombinant protein was studied by exclusion-diffusion chromatography on a 24-mL bed volume Superose 12 column (Pharmacia). The column was equilibrated in 100 m M potassium phosphate, pH 7.5, 150 m M NaCl, at 0.2 mLÆmin )1 . Bovine serum albumin (67 kDa), chicken egg ovalbumin (43 kDa), dimeric bovine b-lactoglobulin (36 kDa), bovine carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21.5 kDa) and bovine ribonuclease A (13.7 kDa), purchased from Sigma, were employed as standards. A 100-lLsampleof purified ASP3c was loaded at 0.5 mgÆmL )1 onto the Superose column and the elution profiles were obtained from on-line UV detection at 280 nm. CD spectra were recorded using a JASCO J-810 spectropolarimeter and analyzed as previously described [42]. ASP3c concentrations were determined using UV spectroscopy employing the extinction coefficient of 11 200 M )1 Æcm )1 at 276 nm, calculated according to Pace et al.[46].Proteinsamples( 1mgÆmL )1 in 50 m M potas- sium phosphate buffer, pH 7.0) were placed in a 0.01-cm path length cell. Baseline was recorded with phosphate buffer. Secondary structure proportions were computed using the algorithm of Deleage & Geourjon [47]. Peptide mapping and disulfide bridge assignment In order to determine the disulfide bridge pairing, ASP3c was digested by trypsin and the resulting peptides were separated by RPLC as described by Briand et al.[48].The fractions were manually collected. N-Terminal amino acid sequence and MALDI-TOF analysis in a reflector mode were performed as described previously. Tryptophan quenching-based ligand binding We tested tryptophan intrinsic fluorescence quenching using brominated fatty acid 15-bromopentadecanoic acid (BrC15-Ac) (Fluka, France) and palmitic acid (C16-Ac). BrC15-Ac and C16-Ac were weighed and dissolved in 100% EtOH as 10 m M stock solutions. Tryptophan fluorescence was determined using an excitation wave- length of 285 nm and an emission wavelength of 326 nm with 1 or 4 l M of ASP3c in 50 m M potassium phosphate buffer, pH 7.5. The concentration of ASP3c was deter- mined using UV spectroscopy as previously described. Spectra were recorded with 4 l M ASP3c at 25 °Cusinga SFM 25 Kontron fluorometer with a 5-nm bandwidth for both excitation and emission. For quenching experiments, successive 0.1-lL ligand aliquots were added to 1 mL of 1 l M ASP3c solution using a 1-lL Hamilton syringe. Dissociation constants (K d ) were calculated from a plot of fluorescence intensity vs. concentration of total ligand, obtained with a standard nonlinear regression method [49] using DELTAGRAPH 4.5 software. Fluorescent fatty acid analogue-based ligand binding Fluorophore ligand binding experiments were performed with 1 l M ASP3c solutions in 50 m M potassium phosphate buffer, pH 7.5. The fluorescent probe (+/–)-12-(9-anth- royloxy)stearic acid (ASA) was obtained from Sigma (France). ASA was dissolved in 10% v/v EtOH as 1 m M stock solution. Successive 0.1-lL ASA probe aliquots were added to 1 mL of ASP3c solution using a 1 lLHamilton syringe. No cut off filter was used in the excitation beam. The excitation wavelength used for ASA was 360 nm. Once the binding equilibrium was reached, in approximately 1 min as verified by time course experiments (not shown), the relative proportion of probe bound to ASP3c was calculated by measuring fluorescence emission (expressed in arbitrary units). Dissociation constants (K d )werecalculated from a plot of fluorescence intensity vs. concentration of total ligand, as described previously. Competitive binding assay The competitive binding assays aimed to displace fluores- cent probe with ligands were performed with 1 l M of ASP3c in 50 m M potassium phosphate buffer, pH 7.5 with 1 l M ASA probe concentration. The synthetic blend correspond- ing to the major components of the queen bee mandibular gland extract was purchased from Phero Tech Inc. (Canada). It is composed of 9-keto-2(E)-decenoic acid (150 lg), 9-hydroxy-2(E)-decenoic acid (71% R-(–), 29% S-(+); 55 lg), methyl p-hydroxybenzoate (13 lg) and 4-hydroxy-3-methoxyphenylethanol (1.5 lg) as defined for one queen equivalent (Qeq), the average amount of pheromone found in the gland of mated queen [50]. The synthetic pheromone blend was dissolved in ethanol to a final concentration of 10 mgÆmL )1 . Other competitor ligands were dissolved in 100% v/v EtOH. In order to prevent solvent competition binding [51], successive 0.1-lL fluorescent probe aliquots were added to 1 mL of ASP3c solution using a 1 lL Hamilton syringe. The EtOH concentration in the binding-assay never exceeded 0.2% v/v leading to a maximum of relative fluorescence decay of 10%. Competitor concentrations causing a fluorescence decay to half-maximal intensity were taken as IC 50 values. The apparent K diss values were calculated as K diss ¼ [IC 50 ]/ (1 + [L]/K d ) with [L] being the free fluorophore concen- tration and K d the OBP-fluorophore complex dissociation constant [52]. RESULTS Cloning of ASP3c In a search of putative soluble proteins involved in chemoreception, we screened a cDNA library prepared from honeybee antennal tissues. One clone encoded for a protein whose N-terminal sequence matched the amino acid sequence determined on ASP3c protein purified from honeybee antennae [20]. Its complete cDNA sequence (Fig. 1) comprises 636 nucleotides, including an open reading frame of 393 nucleotides starting at the ATG codon in position 40 and ending at the TAA codon at positions 430–432. The nucleotide sequence has been 4588 L. Briand et al.(Eur. J. Biochem. 269) Ó FEBS 2002 deposited in the GenBank Sequence Database with acces- sion number AF481963. The open reading frame encodes a 130-amino acid polypeptide. The comparison of the amino acid sequence deduced from the cDNA sequence with that of the N-terminal sequence of the natural ASP3c protein [20] showed that a 21-residue N-terminal signal sequence is cleaved after translation. The average molar mass calculated for the mature protein, assuming the formation of two disulfide bridges, was 12 756.6 Da, in agreement with the measured molar mass (12 758.3 ± 1.7 Da) of the native protein [20]. This protein does not therefore undergo any post-translational modification other than signal peptide cleavage and disulfide bridge formation. The calculated isoelectric point of ASP3c was 5.9, in agreement with those reported for other CSPs. The deduced amino acid sequence of ASP3c compared with those of other insect CSPs and related proteins clearly identified ASP3c as a member of the CSP family (Fig. 2). The honeybee ASP3c protein exhibits 45% to 55% identity with CSP-related proteins from different species, which Fig. 1. Nucleotide and deduced amino-acid sequences of an antennal cDNA clone from Apis mellifera L. corresponding to ASP3c. The nucleotides and amino acids are numbered. The first amino acid of the ASP3c mature sequence, indicated by a vertical arrow, is used as a reference for amino acids numbering. The asterisk marks the stop codon. The disulfide bonds are indicated by a line connecting the circled half-cystines. Fig. 2. Sequence alignment of ASP3c with CSP isoforms and related proteins reported in other insect species. Amino-acid sequences were identified by a BLAST search with ASP3c sequence as a query. Conserved amino acid residues are colored white with black background. Asterisks denote cysteine residues. The conserved tryptophan residue is indicated by an arrow. Representative species are A. mellifera (line 1, EMBL accession code AF481963), S. gregaria (line 2, EMBL accession code AF070962), M. sexta (line 3, EMBL accession code AF117599), P. americana (line 4, EMBL accession code AF030340), L. migratoria (line 5, EMBL accession code AJ251077), D. melanogaster (line 6, EMBL accession code U05244), H. armigera (line 7, EMBL accession code AF368375); M. brassicae (line 8, EMBL accession code AF211180), C. cactorum (line 9, EMBL accession code U95046). Percentage identities of the predicted mature sequence proteins of 8 CSP isoforms with ASP3c are indicated. Ó FEBS 2002 Brood pheromone binding by bee chemosensory protein (Eur. J. Biochem. 269) 4589 indicates a high degree of conservation between CSP-like proteins of phylogenetically distant species. Figure 2 shows that the four cysteines and a single conserved tryptophan are aligned for all known CSP-like sequences. ASP3c heterologous expression Recombinant ASP3c was secreted at high level from the methylotrophic yeast P. pastoris with its natural signal peptide, allowing physico-chemical and functional studies. Samples of expression medium supernatants, taken at various time intervals, were analyzed by SDS/PAGE to determine the optimal induction time. Only the recombinant protein, migrating at approximately 12 kDa, was detectable by Serva blue G staining. The electrophoretic profile (Fig. 3A) reveals the protein regularly accumulating over an expression period of 3 days, while only traces of other proteins were detected. After dialysis of culture supernatant, the recombinant protein was purified by one-step RPLC (Fig. 3B). Recombinant ASP3c eluted as a single peak at 32% acetonitrile just as the natural protein did [20]. Correct processing of the signal sequence was verified by N-terminal analysis of purified ASP3c, demonstrating that honeybee insect signal peptide was efficient for proper secretion of heterologous ASP3c in P. pastoris. MALDI-TOF mass spectrum of recombinant ASP3c (Fig. 4) showed a major peak, together with derivatives corresponding to matrix adducts. The ASP3c mass was found to be 12 757.1 Da, which is in agreement with the theoretical and the measured molecular mass of the natural honeybee protein [20]. The purified ASP3c production reached a level of 17 mgÆL )1 over an expression period of 3 days. Disulfide bridge assignment The recombinant ASP3c protein was subjected to trypsin digestion, which was expected to cleave the polypeptide chain linked by a disulfide bridge. The tryptic peptide mixture was separated by RPLC (not shown) and analyzed by MALDI-TOF mass spectrometry and N-terminal sequencing. The calculated and experimentally determined peptide masses are listed in Table 1. All peptides greater than four residues in length have been identified by mass spectrometry in the chromatogram and, in every case, the measured mass was in perfect agreement with the calculated value. The peptide C29–R35 of mass 823.34 was observed to be linked to the peptide C36–K44 of mass 964.43 resulting in a peptide of 1784.76 (theoretical mass 1784.76), thus demonstrating the existence of a disulfide bond between C29 and C36. Similarly, the peptide V46–K61 of measured mass 1718.84 (theoretical mass 1718.86 with one disulfide bridge) appeared to have an internal disulfide bond between C55 and C58. All three peptides were completely sequenced by automated Edman sequencing. For the peptide C29–R35 linked to peptide C36–K44, two corresponding N-terminal sequences were simultaneously observed in approximately equimolar amounts. For the peptide V46–K61, one conti- nuous sequence was found, despite the fact that three putative trypsin cleavage sites exist within the peptide. The fact that the peptide was not cleaved by trypsin supports the notion of an internal disulfide bond that probably resulted in a compact conformation that was resistant to cleavage. CD analysis and oligomerization The far-UV CD spectrum of ASP3c at neutral pH (Fig. 5A) displayed a positive peak centered at 193 nm and two Fig. 4. MALDI-TOF mass spectrometry analysis of the recombinant ASP3c secreted by Pichia pastoris. Sinapinic matrix adducts are shown. Fig. 3. Electrophoretic analysis and purification of recombinant ASP3c. (A) SDS/PAGE analysis of recombinant ASP3c secreted by Pichia pastoris. Lane 1 shows standards (Low range and Polypeptide kits, Bio-Rad, France) and lanes 2–5 are 50-lL aliquots of 0–3-days culture supernatants. Proteins were visualized by Serva blue G250 staining. (B) Chromatogram of ASP3c purification from the cell culture super- natant by RPLC. Dashed line indicates the acetonitrile gradient. 4590 L. Briand et al.(Eur. J. Biochem. 269) Ó FEBS 2002 negative peaks at 208 nm and 222 nm. This clearly showed the presence of abundant a-helices. The deconvolution of the CD spectrum revealed that ASP3c was composed of approximately 50% a-helix and 5% b-sheet. As shown in Fig. 5B, calibrated exclusion-diffusion chromatography of purified ASP3c at 0.5 mgÆmL )1 exhibited an apparent molecular mass of 15.9 kDa at the sensillar lymph pH of 7.5, which is approximately the value obtained from mass spectrometry (12 757.1 Da), demonstrating monomeriza- tion of the recombinant protein. Binding of ligands assessed by the intrinsic tryptophan fluorescence The recombinant protein appeared therefore quite amena- ble to ligand-binding studies, as it was chemically homoge- neous, with proper conformation, disulfide bridges and secondary structure as expected for a CSP. Intrinsic fluorescent spectroscopy yields information regarding the environment of tryptophanyl residues. ASP3c amino acid sequence (Figs 1 and 2) contains a single conserved tryptophan residue (W81). The fluorescent spec- trum of ASP3c (Fig. 6A) showed a maximum emission of 326 nm, suggesting that this residue is buried within the molecule, possibly involved in the binding site. Lartigue et al. [32] showed that a CSP of M. brassicae is able to bind C12 to C18 alkyl chains. We therefore tested the capability of palmitic acid (C16-Ac) and a fluorescent anthroyloxy derivative fatty acid, ASA to affect ASP3c fluorescence. Upon addition of these compounds, a signi- ficant blue shift of W81 fluorescence emission maximum was observed from 326 to 322 and 315 nm, respectively (Fig. 6A) with a weak increase of fluorescence intensity, showing that ASP3c W81 fluorescence is affected by interaction with these lipophilic compounds. Because some halogenated compounds are known to strongly quench tryptophanyl fluorescence [32,53,54], we measured the interaction of ASP3c with the bromo- substituted fatty acid BrC15-Ac (Fig. 6A). Upon addition of increasing amount of BrC15-Ac, tryptophan fluorescence was strongly quenched (Fig. 6B). The data were fitted by nonlinear regression and the binding constant derived from mathematical analysis was calculated to be 1.7 l M . Fluorescent binding assay using ASA We also directly confirmed the ability of ASP3c to bind the fluorescent probe ASA [55,56]. When excited at 360 nm, ASA presented a weak fluorescence emission with a maximum at 445 nm in aqueous medium (Fig. 7A). In the presence of ASP3c, the maximum underwent an hypso- chrome shift towards 425 nm with a fivefold quantum yield increase. Titration of ASP3c with ASA was saturable (K d ¼ 0.57 l M ) with one binding site per monomer (Fig. 7B). Ligand competitive assays using ASA probe Diverse ligands, representing several classes of chemical structures, were then tested for affinity toward ASP3c in a competitive binding assay with the fluorescent probe, ASA. We first tested MeOH and EtOH, which were used to dissolve ligands and probes. As already reported for rat Table 1. Identification of ASP3c tryptic peptides by MALDI-TOF mass spectrometry. Peptide identification Theoretical mass (M+H) + Measured mass (M+H) + D1–K7 829.36 829.45 F8–R21 1686.81 1686.80 L22–K28 911.50 911.49 C29–R35 linked to 1784.76 1784.76 C36–K44 with a SS bridge V46–K61with an internal SS bridge 1718.84 1718.86 E64–K67 488.31 Not found V69–K71 359.27 Not found F72–K87 1903.00 1903.04 Y88–K93 765.38 765.38 F98–K103 752.35 752.49 L105–V109 515.32 515.30 Fig. 5. Secondary and quaternary structures of the recombinant ASP3c. (A) Circular dichroism spectrum of ASP3c. Protein concentration was approximately 0.5 mgÆmL )1 andpathlength0.01cm(B)Exclusion- diffusion chromatography on Superose 12. The elution positions of the molecular mass standards are indicated by arrows: a, chicken egg ovalbumin (43 kDa), b, dimeric bovine b-lactoglobulin (36 kDa), c, bovine carbonic anhydrase (30 kDa), d, soybean trypsin inhibitor (21.5 kDa), e, bovine ribonuclease A (13.7 kDa). Ó FEBS 2002 Brood pheromone binding by bee chemosensory protein (Eur. J. Biochem. 269) 4591 OBP-1F [51], solvent competition effects were similar with MeOH and EtOH. We used EtOH in subsequent assays because of the greater solubility of lipophilic ligands in this solvent. Although EtOH did quench the fluorescence, the decrease was less than 10%, when the EtOH concentration did not exceed 0.2%. The brominated fatty acid BrC15-Ac, previously shown to quench tryptophan fluorescence of ASP3c, was also found to efficiently compete with ASA for ASP3c binding (Fig. 8). The calculated apparent dissocia- tion constant (K diss ), deduced from the half-maximal inhibition values (IC 50 ), was 0.65 l M . We also compared the influence of fatty acid chain length on ASP3c binding (Table 2). Displacement of ASA was maximal for C16-Ac. It was observed to begin with C14-Ac (K diss ¼ 1.64 l M ), increase with C16-Ac, the best ligand for ASP3c (K diss ¼ 0.51 l M ) and decrease with C18-Ac (K diss ¼ 0.80 l M ). In this series, we included two fatty acid methyl esters (C16-Me and C18-Me), described as compo- nents of brood pheromone [57,58]. They were found to compete with ASA (K diss ¼ 1.02 and 1.23 l M , respectively), but less efficiently than the corresponding nonesterified fatty acids. No binding was found to occur with floral odorants or other components of honeybee pheromones. We assayed 1,8-cineol, 2-isobutyl-3-methoxypyrazine, a-pinene and b-ionone, which are known components of floral scents [59] and 2-heptanone, geraniol, citral, 2-nonanol and isoamyl acetate, known to be honeybee nonsexual phero- mones [40]. Cuticular hydrocarbons (C22 and C30 n-alcanes), involved in nestmate and kin recognition [60], and the synthetic blend corresponding to the major components of the queen bee mandibular gland extract [40] were also unable to displace ASA (not shown). Fig. 6. Binding of fatty acid assessed by intrinsic tryptophan fluores- cence. (A) Fluorescence emission spectra of 4 l M recombinant ASP3c alone (solid squares), in presence of 10 l M palmitic acid (open squares), in presence of 10 l M BrC15-Ac (open circles) and in presence of 10 l M ASA (solid circles). Excitation wavelength was 285 nm and the temperature of the cuvette was maintained at 25 °C. (B) Titration curve of ASP3c with BrC15-Ac; open circles show experimental data, while the solid line is the computed binding curve; excitation wave- length was as in (A), and ASP3c concentration was 2 l M and emission wavelength 326 nm. Fluorescence of ASP3c alone was assigned to 100% in absence of ligand. Fig. 7. Fluorescent binding assay using ASA. (A) Fluorescence emis- sion spectra recorded at 25 °Cof1l M ASA in presence of 1 l M recombinant ASP3c (open squares); solid squares indicate the fluo- rescence of ASA alone (1 l M )andopencirclesthatoftheprotein solution alone (1 l M ). Excitation wavelength was 360 nm (B) Titration curves of ASP3c with ASA; open circles show experimental data, while solid line is the computed binding curve; excitation wavelength and ASP3c concentration were as in (A), emission wavelength was 425 nm; ASA probe formula is inserted. 4592 L. Briand et al.(Eur. J. Biochem. 269) Ó FEBS 2002 DISCUSSION In this work, we have characterized ASP3c, a Hymenop- teran soluble protein found in antennal sensilla of both workers and drones. As previously suggested through N-terminal sequence [20], ASP3c is a novel member of the insect CSP family, on the basis of deduced amino acid sequence similarity and the presence of four cysteines in conserved positions. Amino acid sequence identity among the CSPs from different species is high (45–55%), in contrast to insect PBPs and general OBPs, which are highly divergent. The recombinant ASP3c, expressed using the yeast Pichia pastoris, was found to be identical to the natural honeybee protein according to mass spectrometry and Edman sequencing. Peptide mapping experiments assigned the disulfide pairing (C29–C36 and C55–C58). The same cysteine pairing was exhibited by four CSP isoforms from S. gregaria [23,31]. The high homology between CSPs indicates that disulfide bond pairing Cys(I)–Cys(II) and Cys(III)–Cys(IV) is probably shared by all members of this insect protein family. Because the cysteine residues of the recombinant ASP3c formed only the predicted pair of disulfide bonds, it is likely that the protein was properly folded as corroborated by circular dichroism study. The ASP3c CD spectrum and the secondary structure propor- tions obtained by its deconvolution are similar to those obtained by CD or by NMR analysis with recombinant S. gregaria CSP-sg4[31]andM. brassicae CSPMbraA6 [34]. This suggests a general similar global fold for insect CSPs, which would be composed of six a-helices as observed in the X-ray structure of a CSP from M. brassicae [32]. Like many CSPs from S. gregaria [23], Carausius morosus [26] and M. brassicae [34], honeybee ASP3c was demonstrated to be a monomer by gel filtration at sensillar lymph pH. This monomeric state differs from honeybee PBPs and general OBPs, which were found to be dimeric under natural conditions [39,42]. Although no natural ligand for CSPs has been identified so far, several roles have been proposed for insect CSPs based on their tissue localization. For instance, p10 protein, expressed during leg regeneration in the cockroach Peri- planeta americana, has been proposed to be involved in limb regeneration [29,30]. Because of their localization in anten- nae, tarsi and labrum, it has been hypothesized that the class of CSPs could be involved in CO 2 detection or taste [14]. However, binding of neither radioactively labeled bicar- bonate nor glucose with CSPs of S. gregaria has been observed [23]. Recently, using a fluorescent-binding assay, CSP-sg4fromS. gregaria was observed to bind odorants with a low affinity, whereas carboxylic acids and linear alcohols of 12, 14 and 18 carbons, as well as ethyl esters of the fatty acids, failed to displace the fluorescent probe [61]. However, the structural analogy of CSPs with various transport proteins of lipidic compounds [34] suggested a lipid carrier function possibly involving pheromones or other lipids, such as cuticular compounds. Diverse tritiated pheromonal analogues and fatty acids were observed to bind the CSP of the Lepidopteran M. brassicae [15,17,18]. Moreover, fluorescence quenching and modeling studies showed that the M. brassicae CSPMbraA6 was able to bind brominated alkyl alcohols or fatty acids [32]. The hypothesis of lipid association is well supported by our data. Amino acid sequence alignment revealed that the bee ASP3c contains a single conserved tryptophan residue (W81). Because tryptophanyl residues are frequently involved in ligand binding, the binding of ligands can be monitored by a significant decrease in the intrinsic protein fluorescence due to energy transfer from excited tryptophan residues. Palmitic acid and the fluorescent probe ASA were shown to weakly affect W81 fluorescence. In contrast, among halogenated compounds, which are known to strongly quench tryptophanyl residues [53,54], a bromo- substituted fatty acid, BrC15-Ac was shown to efficiently quench W81, as observed with M. brassicae CSPMbraA6 [32]. We observed also that BrC15-Ac was able to displace ASA, suggesting that brominated fatty acid and ASA both associated with W81 in the same ligand binding site. The ligand binding activity of ASP3c was further investigated using displacement of ASA, a fatty acid probe with an anthroyloxy fluorophore. Anthroyloxy derivatives emission maxima are only weakly affected by solvent polarity. Instead, they are sensitive to rotational steric Fig. 8. Competitive binding assays of ASA with several ligands. EtOH (r), C14-Ac (n), BrC15Ac (m), C16Ac (s), C18-Ac (h), C16-Me (d) and C18-Me (j); fluorescence of ASA-ASP3c complex was assigned to 100% in absence of competitor; experimental conditions were as described in Fig. 6. Table 2. Affinity of ligands for ASP3c measured with ASA as fluores- cent competitive probe. d, maximal percentage of displacement reached at high ligand concentration; IC 50 , ligand concentration provoking a decay of fluorescence of half-maximal intensity; K diss ,apparentdis- sociation constant obtained by K diss ¼ [IC 50 ]/(1 + [L]/K d )with[L]for the free probe concentration and K d the measured dissociation con- stant of ASP3c-ASA complex. Ligand d IC 50 (l M ) K diss (l M ) C14-Ac 27 4.5 1.64 BrC15-Ac 48 1.8 0.65 C16-Ac 58 1.4 0.51 C18-Ac 30 2.2 0.80 C16-Me 45 2.8 1.02 C18-Me 26 3.4 1.23 Ó FEBS 2002 Brood pheromone binding by bee chemosensory protein (Eur. J. Biochem. 269) 4593 hindrance at the level of the anthroyloxy moiety [62]. When bound to ASP3c, the maximal emission wavelength of ASA significantly decreased, revealing rotational constraints and a narrow binding site. Odorants, sexual/nonsexual phero- mones, fatty acids and fatty acid methyl ester derivatives (components of brood pheromone) were tested for their ability to displace ASA. As already observed with CSPM- braA6 [32], we demonstrated that ASP3c was indeed able to bind diverse fatty acids with dissociation constants in the micromolar range with a chemical specificity for aliphatic chains of 16–18 carbons. Methyl ester derivatives were also observed to bind ASP3c, opposite to the S. gregaria CSP, which does not bind lipids either [61]. Moreover, the affinity constant of BrC15-Ac deduced from ASA competition was close to that obtained from tryptophan fluorescence quenching. The only slightly lower affinity of the esterified fatty acid, compared to unsubstituted ones, suggests that the carbonyl group of fatty acids is not essential for binding ASP3c. The micromolar affinity of the tested fatty acids and fatty acid methyl esters for ASP3c is similar to the nanomolar to micromolar binding affinities observed for plant and vertebrate lipid binding proteins [63,64]. More- over, these apparent dissociation constants are very close to those reported for the binding of pheromones and odorants onto insect PBPs and general OBPs [4,42,65], suggesting a physiological role of ASP3c in the transport of fatty acids and their derivatives. 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