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;

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

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-

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

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

)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

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

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

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

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-

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

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

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.

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

]/(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-

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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