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:
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. 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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