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

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,

Characterization of a chemosensory protein (ASP3c) from honeybee

Loı¨c Briand1, Nicharat Swasdipan2, Claude Nespoulous1, Vale´rie Be´zirard1, Florence Blon1,

Jean-Claude Huet1, Paul Ebert2and Jean-Claude Pernollet1

1

Biochimie et Structure des Prote´ines, Unite´ de recherches INRA 477, Jouy-en-Josas Cedex, France;2Department 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

insect species, which are believed to be involved in chemical

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-pheromone-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)

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

E X P E R I M E N T A L P R O C E D U R E S

Strains and materials

Escherichia colistrain 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 MGWBiotech

(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 105

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 withCLUSTAL Wusing 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 ofPichia 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 mMEDTA 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 4C, 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 mM 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 mM ammonium acetate, pH 7.0, 60% v/v acetonitrile in H2O) 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

H2O 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 lL of 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

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 mMpotassium phosphate,

pH 7.5, 150 mM 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-lL sample of

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 200M )1Æcm)1at 276 nm, calculated according to Pace

et al [46] Protein samples (
1 mgÆmL)1in 50 mM

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 mM stock solutions Tryptophan

fluorescence was determined using an excitation

wave-length of 285 nm and an emission wavewave-length of 326 nm

with 1 or 4 lM of ASP3c in 50 mMpotassium phosphate

buffer, pH 7.5 The concentration of ASP3c was

deter-mined using UV spectroscopy as previously described

Spectra were recorded with 4 lMASP3c at 25C using a

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 lM ASP3c solution using a 1-lL Hamilton syringe

Dissociation constants (Kd) were calculated from a plot of

fluorescence intensity vs concentration of total ligand,

obtained with a standard nonlinear regression method [49]

Fluorescent fatty acid analogue-based ligand binding

Fluorophore ligand binding experiments were performed with 1 lMASP3c solutions in 50 mMpotassium 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 mM stock solution Successive 0.1-lL ASA probe aliquots were added to 1 mL of ASP3c solution using a 1 lL Hamilton 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 (Kd) were calculated 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 lMof ASP3c

in 50 mMpotassium phosphate buffer, pH 7.5 with 1 lM 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 IC50values The apparent Kdissvalues were calculated as Kdiss¼ [IC50]/ (1 + [L]/Kd) with [L] being the free fluorophore concen-tration and Kdthe OBP-fluorophore complex dissociation constant [52]

R E S U L T S

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

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.

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.

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 lM 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 (Kd¼ 0.57 lM) 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) +

C36–K44 with a SS bridge

V46–K61with an internal

SS bridge

Fig 5 Secondary and quaternary structures of the recombinant ASP3c.

(A) Circular dichroism spectrum of ASP3c Protein concentration was

approximately 0.5 mgÆmL)1and path length 0.01 cm (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).

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 (Kdiss), deduced from the half-maximal

inhibition values (IC50), was 0.65 lM 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 (Kdiss¼ 1.64 lM),

increase with C16-Ac, the best ligand for ASP3c

(Kdiss¼ 0.51 lM) and decrease with C18-Ac (Kdiss¼ 0.80 lM) 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 (Kdiss¼ 1.02 and 1.23 lM, 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 C of 1 l M ASA in presence of 1 l M

recombinant ASP3c (open squares); solid squares indicate the fluo-rescence of ASA alone (1 l M ) and open circles that of the protein 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.

D I S C U S S I O N

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] and M 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 CO2 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-sg4 from S 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 perfluores-centage of displacement reached

at high ligand concentration; IC 50 , ligand concentration provoking a

decay of fluorescence of half-maximal intensity; K diss , apparent

dis-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.

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 Interestingly, ASP3c efficiently binds

fatty acid ester components of the brood pheromone,

although it was found to be unable to bind nonfatty acid

general odorants and other tested pheromones (sexual and

nonsexual) This pheromone is known to be involved in the

regulation of behavioral sequence including feeding of

the larvae, capping of the cells and thermoregulation of the

brood area in the colony

The recombinant protein ASP3c is produced in sufficient

quantity to provide enough material for the crystallization

trials, which are currently under way Moreover, we expect

to locate the binding sites using site-directed mutagenesis

aiming to clearly define the relationships between the

structure and the function of this honeybee CSP

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