Chem. Senses 27: 691-701,
2002
© Oxford University Press 2002
Porcine Odorant-binding Protein Selectively Binds to a Human Olfactory Receptor
Laboratoire de Neuroglycobiologie, IFR du Cerveau, GLM-CNRS, 31 Chemin J. Aiguier, F-13402 Marseille cedex 20, France 1 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 660 route des Lucioles, Sophia Antipolis, F-06560 Valbonne, France 2 Institute of Food Research, Norwick Research Park, Colney, Norwich, UK 3 Institut de Génétique Humaine, CNRS UPR 1142, rue de la Cardonille, Montpellier, France
Correspondence to be sent to: Catherine Ronin, Laboratoire de Neuroglycobiologie, IFR du Cerveau CNRS, 31 Chemin J. Aiguier, F-13402 Marseille cedex 20, France. e-mail: ronin{at}irlnb.cnrs-mrs.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (Kd = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (Bmax = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The sense of smell allows the perception and discrimination of thousands of odorant molecules in living organisms from invertebrates to mammals. Such chemical signalling modulates the social behaviour of most animal species which rely on odorant compounds to identify kin, mate, to locate food or to recognize territory. Olfactory perception is based on the activation by odorant molecules of G-coupled receptors, designed as olfactory receptors (ORs) and located at the cilia of olfactory neuronal endings (Breer, 1994
). Odorants, which
are commonly hydrophobic molecules, have to be conveyed from the air through
the aqueous nasal mucus by carriers. Odorant-binding proteins (OBPs) have been
thought to play such a functional role
(Pelosi, 1996). These
low-molecular soluble molecules are similar to retinol-binding protein and
belong to the lipocalin family [for review, see Flower
(Flower, 1994)]. They are
physiologically secreted in high amounts in the nasal mucus layer [for review,
see Flower (Flower, 1996)]
but, despite a putative odorant carrier function, their precise role in early
olfactory perception remains unclear
(Tegoni et al.,
2000). In particular, a potential recognition between OBP and OR
has not yet been clearly established and therefore could be an important key
in the understanding of this complex mechanism.
Olfactory receptors are encoded by several hundreds of genes in mammals,
possibly representing 3% of their genome
(Mombaerts, 2001). Full or
partial OR sequences have been cloned from genomic DNA or olfactory epithelium
cDNA in humans and in other vertebrates (dog, pig, rodents, chicken, catfish)
as well as in invertebrates (C. elegans and Drosophila) [for
review, see Mombaerts (Mombaerts,
1999)]. Interestingly, OR expression is not restricted to the
olfactory epithelium but has also been observed in other tissues like testis
(Parmentier et al.,
1992; Thomas et al.,
1996), notochord (Nef and Nef,
1997), insulin-secreting beta cells, spleen and heart
(Blache et al., 1998),
where their function remains to be deciphered. Pioneering studies have
attempted to elucidate OR function by expressing gene sequences in either
insect or mammalian cells and screening for odorant signalling
(Raming et al., 1993;
Krautwurst et al.,
1998; Wetzel et al.,
1999). Specific recognition of odorant molecules has been shown to
be lilial and lyral for rat OR 5 receptor
(Raming et al.,
1993), heptanal for mouse OR 17
(Krautwurst et al.,
1998) and helional for human OR 17-40
(Wetzel et al.,
1999). Even though heterologous OR expression appeared to suffer
from limited cell surface targeting, these data formally demonstrated a direct
recognition of the odorant ligand by the receptor that occurred in the absence
of an OBP. More recently, a survey of mammalian OR specificity based on a set
of structurally related odorants demonstrated that a single OR could recognize
different odorant molecules sharing the same odotope and that a single odorant
is recognized by multiple ORs (Malnic
et al., 1999). Such a broad recognition mechanism in
perception events suggests the existence of a combinatorial receptor coding
scheme to encode odor identities, contributing to olfactory
discrimination.
The cascade of perireceptor events encompassing the recognition of odorant
by the intermediate of a carrier protein has also remained very elusive. OBPs
are very abundant in the olfactory mucus, suggesting that these molecules may
transport odorants to ORs (Pevsner et
al., 1985). Bovine OBP (bOBP) was first discovered for its
ability to bind 2-isobutyl-3-metoxypyrazine (IBMP), described as `bell pepper
smell' (Pelosi et al.,
1982). Its crystallographic structure consists of an homodimeric
protein, exhibiting a domain-swapping mechanism and containing an endogenous
ligand in its internal binding cavity
(Bianchet et al.,
1996; Tegoni et al.,
1996). Recently, the X-ray structure of pOBP has been also
determined (Spinelli et al.,
1998). Despite the fact that pOBP and bOBP are folded in the
typical ß-barrel lipocalin structure, pOBP is monomeric and devoid of
naturally occurring bound ligand. However, a conformational stability study of
pOBP further revealed the existence of a monomer—dimer equilibrium
depending on experimental conditions
(Burova et al.,
1999).
Interestingly, a wide variety of odorant compounds were shown to bind to
bOBP (Pevsner et al.,
1990) as well as pOBP, within a micromolar range of affinity. It
has been recently demonstrated that odorants of unrelated chemical structure
can bind to pOBP with similar affinities by interacting with different amino
acid residues in the binding pocket
(Vincent et al.,
2000). As a result, OBPs may trap unrelated odorants and function
as scavengers for lipophilic ligands [reviewed by Pelosi
(Pelosi, 1994)]. Therefore,
OBP is currently thought to exert its function in vivo by regulating
odorant delivery across the hydrophilic mucosal barrier to the membrane
receptors located at the cilia tips of the olfactory neuron dendritic knob. A
role for OBP in odorant removal to terminate the odorant response has also
been proposed (Boudjelal et al.,
1996). In this case, OBPs would be involved in a detoxification
role, common to several members of the lipocalin family, and would thereby
prevent receptor desensitization. To assess further such a dual role for OBPs,
it would be of importance to determine whether these mechanisms occur
sequentially or are controlled by binding specificity and odorant
concentration at the OR level.
Low-affinity binding sites for bOBP have been described in nasal and
respiratory epithelia (Boudjelal et
al., 1996), supporting the hypothesis that OBP binds outside
the olfactory tissue, but evidence that these sites account for ORs has not
yet been ascertained. Our understanding of the molecular interaction of
odorants with OBPs and ORs is thus rather limited at a time when molecular
cloning of ORs and genome sequencing are providing us with hundreds of orphan
genes which require functional identification. In this study, we addressed a
putative OBP—OR recognition by taking advantage of known full-length
gene sequences of ORs from human genome. Among the 16 OR genes present in the
locus p13.3 of chromosome 17, we first identified two orphan genomic sequences
as being expressed specifically in human nasal epithelium. Using purified
radiolabelled porcine OBP and genetically engineered human ORs, we have been
able to show a differential pOBP binding to the receptors. To our knowledge,
these data provide the first evidence of a selective recognition between OBP
and OR. Interestingly, this interaction was found to be of high affinity,
suggesting that the OBP—OR complex may very well occur under
physiological conditions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Animals and chemicals
BALB/c mice were obtained from IffaCredo (Orléans, France) and were
housed in a temperature-, humidity- and light-controlled room with free access
to food and water. The pOBP (also called pOBP-I, major component of porcine
nasal mucus) was kindly provided by Professor Paolo Pelosi. The protein was
obtained and purified according to the procedure described by Dal Monte et
al. (Dal Monte et al.,
1991). Molecular biology products (restriction enzymes and
synthetic oligonucleotides) were purchased from Eurogentec Belgium; polymerase
enzymes from Roche Molecular Biochemicals; polynucleotide kinases from
Promega; human testis cDNA and plasmids from Clontech. Cell culture reagents;
DMEM, fetal calf serum, trypsine/EDTA, geneticin, gentamicin and fungizone
were purchased from Invitrogen. Biochemical products were from Sigma-Aldrich
(1, 10-phenantroline), ICN Biomedicals ([125I]Na) and Calbiochem
(lactoperoxidase).
Expression of ORs in human olfactory epithelium and testis
Genomic DNA was isolated from blood cells (kindly provided by Dr Francis
Castet, CNRS, Marseille). Olfactory cDNA library was prepared from human
middle turbinate tissue using the Stratagene vector lZAPII
(Crowe et al., 1996).
Specific internal primers for PCR amplification and for sequencing were
designed to amplify specific OR genes from a human chromosome 17 cluster
(17p13.3), based on the available sequence
(Rouquier et al.,
1998) as follows: OR 17-201, 5'-CTCTTGTCCCACAAGTCC-3'
and 5'-TTATCCTTGTCTGAAA-3' (370 bp); OR 17-209,
5'-GACTGCTACGTGGGCATA-3' and 5'-TGTGAGCTGCAGGTGGAA-3'
(373 bp); OR 17-210, 5'-ATGGCTTATCACTGCTAT-3' and
5'-GTGGAGAAAGCCTTCTGG-3' (370 bp). One nanogram of DNA was used in
each of a series of polymerase chain reaction (PCR) experiments containing 10
mM Tris—HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001%
gelatin, 5 mM dNTP, 1.5 U of Taq DNA polymerase (Boehringer-Mannheim) and 100
pmol of each primer. PCR mixtures using the olfactory or testis cDNA (Human
testis Quick-CloneTM cDNA, Clontech) library has been carried out in the
presence of 3mM MgCl2 according to the following schedule: 94°C
(90 s, 1 cycle), 94°C (20 s), 50°C (25 s), 72°C (30 s, 40 cycles)
and 72°C (120 s, 1 cycle). PCR reactions were analysed by gel
electrophoresis in 1% agarose, further purified and sequenced to verify the
specific amplification of ORs.
Construction of p-EGFP/OR 17-209 and p-EGFP/OR 17-210
The intronless genes encoding OR 17-209 and OR 17-210 putative olfactory receptors from human chromosome 17 (p13.3) were amplified by PCR using the cosmid no. ICRF105cF06137 (GenBank HSU53583). In order to create a GFP C-terminal fusion protein, OR 17-209 and OR 17-210 genes were mutated to eliminate the stop codon. The substitution of the stop codon with a BamHI restriction site allowed in-frame subcloning of the new sequence with GFP cDNA, in the pEGFP-N1 mammalian expression plasmid (Clontech). The following oligonucleotidic sense (S) and antisense (AS) primers were used (Eurogentec): OR-209 HindIII S: 5'-TAA GAA GCT TGC CAC CAT GGA GGG GAA AAA TCT G-3'; OR-209 stop/BamHI: 5'-ATT AGG ATC CCC AGG GGA ATG AAT TTT CCG-3'; OR-210 HindIII S: 5'-CTG TAG GTG TTA AGG TGC ATT-3'; OR-210 stop/BamHI: 5'-ATT AGG ATC CCC AGC CAC TGA TTT AGA GTG-3'. PCR experiments containing 10 ng of cosmid DNA preparation, 10 mM Tris—HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTP, 1.5 U of high-fidelity ExpandTM mix of polymerases (Roche Molecular Biochemicals) and 100 pmol of each primer were performed according to the following schedule: 94°C (90 s, 1 cycle), 94°C (20 s), 50°C (25 s), 72°C (90 s, 40 cycles) and 72°C (120 s, 1 cycle). The pEGFP-N1 recombinant clones were isolated using HindIII/BamHI restriction enzymes (Eurogentec) and were fully sequenced to assess the expected inserted DNA sequence.
Construction of pS 5HT1C
The membrane import sequence of the human 5H-T1C receptor was synthetically reconstituted. Four sense oligonucleotides and four antisense oligonucleotides corresponding to the complete 5H-T1C membrane import sequence were synthesized. One microgram of each internal oligonucleotides was phosporylated using 1 U of polynucleotide kinase (Promega) in a kinase buffer containing 2 mM of ATP. Reaction was performed for 60 min at 37°C, and the incubation finally inactivated for 10 min at 65°C. All the oligonucleotides were denatured for 10 min at 80°C and then mixed for matching. Matching was performed overnight with a decreasing temperature gradient from 80 to 20°C. The resulting fragment was then ligated into the Bgl2 and HindIII sites of the pEGFP-N1 plasmid in frame with the N-terminal OR sequences.
Cell culture and transfection
COS-7 cells (ATCC reference: CRL-1651) were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum and containing 50 µg/ml gentamicin and 2.5 µg/ml fungizone, in a humidified incubator (95% O2, 5% CO2) at 37°C. Stable transfection (with either 5HT1C-OR-209-GFP or 5HT1C-OR-210-GFP construct) was performed onto semiconfluent COS-7 cells grown in 100 mm cell culture dishes with 2 µg of recombinant pEGFP-N1 plasmid using the lipofectAMINETM (Gibco) reagent according to the manufacturer's recommandations. After 2 days, cells were cultivated with 0.5 g/l of geneticin.
Confocal microscopy
All fixation steps were performed on ice to prevent receptor internalization. COS-7 cells were fixed in 3% paraformaldehyde buffer for 20 min. Fixed cells were washed with 0.05 M NH4Cl for 10 min, and then twice with PBS for 5 min. Cells were fixed to slides with Mowiol (Vector) and dried for 12 h. Images of fluorescent (GFP) cells were obtained using a Leica TCS 4D microscope with excitation at 488 nm.
Iodination of pOBP
The pOBP was submitted to iodination using the lactoperoxidase method
(Marchalonis, 1969). Briefly,
2 nmol of purified pOBP in 50 mM borate/phosphate buffer (pH 7.5) were
incubated with 2 mCi of [125I]Na, 10 µg of lactoperoxidase and
12 µl of H2O2 (100% diluted 800 times). After 15 min
of incubation, the reaction was stopped by dilution in 0.1% trifluoroacetic
acid (TFA). The iodination mixture was purified by HPLC onto a nucleosil
column (C18, 300 A, 5 µm) eluted at a flow rate of 1 ml/min in 0.1% TFA
with an initial 5 min isocratic 20% acetonitrile step, followed by a 45 min
linear gradient from 20 to 60% acetonitrile. The chromatography was followed
by automatic recording of absorbance at 275 nm and radioactivity. Fractions
were collected into low absorption tubes, diluted in 50 mM Tris—HCl
buffer (pH 7.5), containing 10% BSA, aliquoted and stored at -20°C until
use.
SDS—PAGE analysis of the radiolabelled pOBP fractions
Fractions of each radiolabeled pOBP (25 000 c.p.m.) were heated at 95°C
for 5 min in the presence of 5% of ß-mercaptoethanol. Samples were
submitted to SDS—PAGE analysis according to the procedure of Laemmli
(Laemmli, 1970) on a 14%
polyacrylamide resolving gel. The coloured wide-range prestained protein
marker from Sigma was used as a standard. The gel was fixed for 10 min in 10%
(v/v) acetic acid and dried under vacuum before radioautography on Kodak
X-OMAT AR film for 1 week.
Binding of 125I-OBP on cells expressing OR 17-209 or OR 17-210 receptors
Untransfected cells and transfected cells, with either 5HT1C-OR-209-GFP or 5HT1C-OR-210-GFP construct, were grown in 24-well plates until confluence, and were washed twice with 0.5 ml of binding buffer (Earle—HEPES buffer pH 7.4, supplemented with 0.09% glucose and 2% BSA) and then incubated for 15 min at 37°C in the same buffer. Association kinetics were followed by measuring the binding of 0.4 nM 125I-labelled OBP to OR-expressing cells at different times. After 60 min of association, the dissociation kinetic was initiated by adding 10 µM unlabelled pOBP to the incubation medium. In saturation experiments, whole cells were incubated for 25 min at 37°C with increasing concentration of 125I-labelled OBP (0.05-18.4 nM). At the end of the incubation period, cells were then washed twice with 0.5 ml of ice-cold binding buffer and harvested in 1 ml 0.1 M NaOH. The radioactivity bound to the cells was measured with a gamma counter (Packard, counting efficiency 80%). Non-specific binding was measured in the presence of a excess of unlabelled OBP (10 µM) and substracted from total binding to obtain specific binding. No specific binding was observed on untransfected COS-7 cells (data not shown). ka, kd and Kd (ka/kd) values were calculated as previously described (Dal Fara et al., 2000).
Binding and radioautography on whole mouse slice
Slide-mounted sections of newborn (P15) BALB/c mice (IffaCredo,
Orléans, France) were prepared as previously described
(Gaudriault et al.,
1994). Preincubation was performed for 15 min at 4°C in a 50
mM Tris—HCl buffer (pH 7.4) containing 5% BSA and 0.8 mM
1,10-phenantroline (Sigma-Aldrich), a metalloprotease inhibitor. Slices were
incubated with 2 x 106 c.p.m. (1.4 nM) of radiolabelled pOBP
in a 400 µl final volume of the same buffer for 30 min at room temperature.
Non-specific binding was determined in the presence of an excess (10 µM) of
unlabelled pOBP. Sections were then washed three times for 5 min in the
ice-cold buffer containing 2% BSA and 0.8 mM 1,10-phenantroline, and finally
washed in ice-cold distilled water (without BSA) for 5 min. Slide-mounted
sections were apposed to a beta Max Hyperfilm (Amersham) in a Kodak X-ray film
cassette for 8 weeks at -70°C.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Screening cDNA libraries for OR expression
Putative olfactory sequences reported from earlier studies have been cloned
from human testis (Parmentier et
al., 1992) while most available OR sequences have been
deduced from genomic DNA. Many of them have been located in clusters, and of
these, the p13-3 locus of chromosome 17 has been extensively studied
(Ben-Arie et al.,
1994; Rouquier et
al., 1998). Three human genes distributed in this locus and
encoding putative olfactory receptors (OR 17-201, OR 17-209 and OR 17-210)
were selected to initiate this study. Their full-length sequence was
intronless like all other mammalian OR genes reported so far, and presented an
open reading frame suited for heterologous protein expression.
Since no information was available on physiological expression of these
genes, we first investigated the presence of such sequences in cDNA libraries
of both human olfactory epithelium and testis. Using specific primers designed
to map the whole set of ORs located in the locus 17p13.3, we showed that OR
17-209 and 17-210 are actually expressed in the olfactory library, described
elsewhere (Crowe et al.,
1996) but not in the testis library. Indeed, gel analysis
(Figure 1) of the PCR products
revealed amplified material for OR 17-209 and OR 17-210 only in the olfactory
cDNA library with a single band at the expected size (370 bp). Sequencing PCR
products revealed a single sequence per band corresponding to OR 17-209 and OR
17-210 respectively, while OR 17-201 could not be identified. Of note, OR
17-24 and OR 17-40 were also identified in the olfactory library. Since these
latter could also been found in testis
(Vanderhaeghen et al.,
1997), our findings further support the view of a differential
expression of the OR repertoire in the olfactory epithelium.
|
Both OR 17-209 and OR 17-210 receptors have not yet been characterized for
biological function and odorant recognition. Their protein sequences share 45%
homology and showed a very distinct hydropathy profile with seven putative
transmembrane domains (data not shown). When aligned with bovine rhodopsin,
whose structure has been recently elucidated
(Palczewski et al.,
2000), OR 17-209 showed 50% identity with the photoreceptor while
OR 17-210 exhibited only 36% identity with a remarkably short N-terminus.
These data strengthen the bioinformatic analysis indicating that both proteins
belong to two distinct OR subfamilies.
Heterologous expression of OR 17-209 and OR 17-210
Plasmids containing full-length OR sequences were then constructed for
protein expression in mammalian cells. Earlier work from another group
reported that expression of ORs at the surface of host cells is limited by
unknown factors (Gimelbrant et
al., 1999). A significant improvement could be observed by
adding an heterologous import signal peptide to address receptor biosynthesis
to the endoplasmic reticulum and facilitate proper intracellular sorting
(Wetzel et al.,
1999). Stably transfected COS-7 cells expressing the OR 17-209 and
OR 17-210 gene sequences fused with the GFP protein revealed abundant
fluorescent protein material, apparently addressed to the perinucleus region
of the cells and probably retained in the endoplasmic reticulum
(Figure 2A,C). To address OR
proteins to the plasma membrane, we took advantage of the 5H-T1c
signal peptide, reported to facilitate intracellular trafficking of unrelated
receptors (Wetzel et al.,
1999). As shown in Figure
2B,D, the presence of such heterologous peptide remarkably shifted
the routing of OR chimeric proteins toward the cell surface. In addition to
the labelling of intracellular compartments, numerous clusters of fluorescent
patches could be observed at the cell surface and in the periplasmic space,
indicating that both receptor proteins now followed a more efficient migration
within the cell. Since no import sequence is originally present in both OR
genes, it is likely that in olfactory neurons, some accessory mechanism must
be involved for these proteins to reach the dentritic knob endings
(Gimelbrant et al.,
2001).
|
Iodination and purification of radiolabelled pOBP
According to amino acid sequence analysis
(Paolini et al.,
1998), five tyrosine residues are potentially available for
iodination. To prevent protein denaturation, derivatization of pOBP
preparation was carried out under experimental conditions limiting iodine
supply, in order to obtain a single iodinated tyrosine residue per molecule.
Three distinct labelled products were thereby obtained and separated by
reverse-phase HPLC (Figure 3).
The ratio of one iodine atom per mole of protein was assessed in each fraction
by optical density coupled to radioactivity measurements. This labelling
procedure was reproducible, and monoiodination of the ligand was achieved in
consecutive experiments. Purified, radioiodinated products were further
analysed by gel electrophoresis followed by autoradiography. Two protein bands
of apparent mol. wt 22 and 44 kDa were identified at the expected molecular
size for monomeric and homodimeric pOBP respectively
(Figure 3, inserts). Both
protein forms were present in each fraction with the monomeric form
significantly predominant over the dimeric form. Since gel analysis was
carried out under denaturing conditions, it appeared that pOBP dimers can
partially resist reduction by forming strong hydrophobic interactions, a
molecular feature reported by Burova et al.
(Burova et al.,
1999).
|
Binding experiments
In preliminary experiments, we tested [125I]OBP binding to
membranes isolated from COS-7 cell clones expressing either OR 17-209 or OR
17-210. Isolating radiolabelled membranes onto various filters in the presence
or absence of polyethylenimine always led to a high, non-specific binding, and
therefore filtration procedures were not retained for further binding assays
(data not shown). Instead, experiments performed on whole cells revealed a
much lower level of non-specific binding. It has been recently observed that
tagging another olfactory receptor (OR-17) by GFP located in C-terminal
position does not affect ligand binding or downstream signaling
(Ivic et al., 2002).
Figure 4A shows that
radiolabelled peaks 1 and 2 did not exhibit any detectable interaction with OR
17-209 or OR 17-210 transfected cells, indicating that these two iodinated OBP
fractions probably lost their binding activity upon derivatization. In
contrast, fraction 3 was found to bind to a much higher extent to OR 17-210
expressing cells compared to the OR 17-209 clone or untransfected cells
(Figure 4A). Binding of the
tracer could be totally displaced with a micromolar concentration of
unlabelled OBP, thereby demonstrating that the iodinated product and the
native protein display similar recognition of OR 17-210. This fraction was
likely to contain an active iodinated form of pOBP and was therefore selected
for binding experiments.
|
) and dissociation (
) of [125I]pOBP (0.4 nM) to OR
17-210 expressing cells (160 µg protein/well). After 60 min of association
at 37°C, 10 µM of unlabelled pOBP was added (arrow). Inserts:
Pseudo-first order representation of data for both kinetics (
We then examined the kinetic parameters of [125I]OBP interaction
with OR 17-210 expressing cells. The association kinetics shown in
Figure 4B indicated that
[125I]OBP specific binding was time-dependent and reached an
apparent equilibrium at 
30 min with a half-maximal binding at 4-6 min.
After 60 min of association, [125I]OBP binding could be reversed by
addition of 10 µM of unlabelled OBP to the cells. Half-maximal release of
the tracer was also 5 min. The calculated rate constants for association
(ka) and dissociation (kd) were about
8 and 7 pM-1 min-1 respectively. These kinetic constants
allowed us to determine an apparent Kd
(kd/ka) of 8.77 nM.
As shown in Figure 5, saturation experiments demonstrated that the specific binding of OBP to OR 17-210 expressing cells was saturable. Scatchard analysis of binding data revealed a single population of high-affinity binding sites with an apparent Kd of 9.5 nM and a maximal binding capacity of 66 fmol/mg prot. Therefore, Kd values deduced from kinetic experiments (Figure 4B) and saturation experiments (Figure 5) were in good agreement. Successive preparations of iodinated pOBP show similar results to those represented in Figures 4 and 5.
|
Mass spectrometry was also performed in non-denaturing condition in order to ascertain whether our pOBP preparation contained any endogenous ligand. The protein was given a molecular mass of 17 690.27 ± 0.61 Da, which is in good agreement with the molecular mass deduced from the pOBP sequence. Porcine OBP preparation was also capable of binding odorant since its size increased after incubation with IBMP (S. Canarelli and O. Clot-Faybesse, unpublished data). It was thus concluded that pOBP was unliganded and functional. These findings demonstrate also that interaction between OBP and OR can physiologically occur in the absence of odorant.
Expression of [125I]pOBP binding sites in mouse tissues
To further comfort the biological activity of the iodinated pOBP and potentially to assess binding sites in other tissues, we performed in situ binding on whole postnatal mice saggital sections. Slices were incubated with each of three radiolabeled pOBP fractions and non-specific binding was measured by displacing the tracer with 10 µM of unlabeled pOBP. As shown in Figure 6, only fraction 3 exhibited specific binding in these experiments. Non-specific binding was very weak with fraction 3 and specific binding was thus comparable to total binding. Again, fractions 1 and 2 failed to present any significant physiological binding, indicating that iodination resulted in a loss of activity for these two forms. In contrast, a strong signal was present with fraction 3 at the level of nasal fossa turbinates, as expected for a ligand that should bind to ORs located in the olfactory epithelium. When autoradiography was carried out at intermediate time intervals, the labelling intensity of nasal cavity was found to reach saturation rapidly, while the labelling of regions outside the nasal epithelium increased consistently. A 2 month exposure (Figure 6) allowed the visualization of specific peripheral binding in the following regions: nasal epithelium, tracheal, intervertebral, intercostal and hit joint cartilages, liver, stomach and intestinal surface, thymus, dorsal muscles, heart and possibly coronaries. These findings show that pOBP preferentially binds to nasal fossea but can also bind to several other regions, suggesting that various tissues may express secondary binding sites for this protein.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Odor perception occurs as a result of a cascade of biochemical and electrophysiological reactions in which OBP and OR molecules are key partners in odorant uptake. Despite a general agreement that OBPs are important to the signalling process, clear experimental evidence of their role in olfactory perception has not yet been produced and their precise function still remains elusive. In a attempt to elucidate if they are part of these early events, we decided to investigate a putative OBP—OR interaction using cell lines that express human ORs.
OR gene sequences are found at >25 locations in the human genome
covering all chromosomes except chromosomes 20 and Y
(Rouquier et al.,
1998). OR clusters have been identified on several chromosomes and
100 genes have been partially or fully sequenced. So far, this large
repertoire of protein receptors has not yet been fully elucidated for
expression and specificity. Located on chromosome 17, one cluster of 16 OR
genes, designated as the 17p13.3 locus, has been studied in considerable
detail by several groups, including one of ours
(Ben-Arie et al.,
1994; Rouquier et
al., 1998; Sharon et
al., 1999). In this cluster, OR genes were found to be
distributed among two families and are thought to originate from duplication
mechanisms. A large part (72%) of total human OR sequences, however, appeared
to be pseudogenes (Rouquier et
al., 1998). Since it has been previously reported that a
17-40 OR gene transcript has been found to be expressed in adult nasal
epithelium (Crowe et al.,
1996), it was of importance to determine for each OR originating
from the human 17p13.3 cluster whether or not they also potentially display
the same location. PCR screening in both adult nasal tissue and testis showed
that OR 17-209 and OR 17-210 were found to be specifically present in an
olfactory cDNA library. This suggested to us that both gene transcripts were
putative candidates for odorant recognition. Using a chimeric construction, we
have been able to obtain OR proteins successfully expressed at the surface of
host cells and could thereby engineer stable cell lines.
To show a potential interaction between OBP- and OR-expressing cell lines,
an OBP partner had to be selected at a time when no human OBP was cloned and
thus available for homologous binding. We hypothesized that heterologous
OBP—OR binding could happen, provided receptor recognition was an
intrinsic function of OBP. This hypothesis was based on the observation that
amino acid sequences are more conserved within OBPs from the same class, but
different animal species, than between proteins of the same species [for
review see Pelosi (Pelosi,
1996)], suggesting that structural differences of OBPs could be
related to odorant-binding specificities, not to OR recognition. pOBP was an
excellent candidate to address this issue because it was available in a highly
purified form, its binding properties have been extensively studied
(Buroa et al., 1999;
Vincent et al.,
2000), and both its sequence
(Paolini et al.,
1998) and its structure
(Spinelli et al.,
1998; Perduca et al.,
2001) have been determined. The X-ray crystallography of pOBP
(Spinelli et al.,
1998) revealed that this protein is monomeric and is devoid of
naturally occurring bound ligand. In contrast, recent work based on the
crystal structure of a monoclinic form of pOBP revealed the presence of an
endogenous ligand (Perduca et
al., 2001). This discrepancy could be accounted for by the
protocol used to purify pOBP from porcine mucus. An additional stage of
extraction with organic solvent is needed to eliminate potential endogenous
ligand in the binding cavity of the pOBP sample
(Burova et al., 1999).
In this study, we assessed by mass spectrometry that the pOBP preparation used
for binding studies was devoid of ligand.
Based on sequence analysis, pOBP contains five tyrosine residues (Tyr20, Tyr52, Tyr78, Tyr82 and Tyr92) among the 157 amino acid residues. Upon monoiodination of the protein, three fractions differing in hydrophobicity were obtained, indicating that not all five tyrosine residues were accessible to derivatization in our experimental procedure. The two major labelled species (fractions 1 and 2) displayed no binding activity towards OR 17-209 or OR 17-210, while the minor peak (fraction 3) exhibited selective interaction with OR 17-210. These findings suggest that at least two tyrosine residues of pOBP are essential for receptor recognition. Regarding fraction 3, iodination is likely to occur on a residue located distally from the OR binding site to generate to an active tracer.
In situ binding studies in mice further strongly supported the
evidence of an heterologous recognition between OBP and target-binding sites.
They confirmed the olfactory epithelium as the major target tissue for pOBP
but also showed secondary binding sites. In mice, we found specific binding
sites in many other locations such as tracheal, intervertebral, intercostal,
hit joint cartilages, liver, stomach and intestinal surface, thymus, dorsal
muscles, heart and coronaries. It is worth noting that OR expression has been
also documented in many tissues outside the olfactory epithelium, e.g. germ
cells and testis (Parmentier et
al., 1992; Thomas et
al., 1996), notochord
(Nef and Nef, 1997), spleen
and insulin-secreting ß-cells (Blache
et al., 1998), heart
(Drutel et al., 1995;
Ferrand et al., 1999)
and possibly in distinct brain areas
(Raming et al.,
1998). Taken together, these findings suggest that the observed
labelling of regions outside the olfactory epithelium may very well reflect
the presence of peripheral OBP-binding sites. Since tissue specificity is of
major relevance when defining the physiological role of a protein, the
presence of ORs in these regions remains to be identified by in situ
hybridization techniques. It would be also of interest to follow the
distribution of these binding sites during ontogenesis to understand if OBP
binding sites are dependent upon development.
Earlier work using radiolabelled bovine OBP (bOBP) (which like pOBP
contains five tyrosine residues) showed binding to isolated membranes from
olfactory and respiratory epithelium with an apparent Kd
value of 2 µM (Boudjelal et
al., 1996). Because such a low-affinity interaction is not
encountered in the G-coupled receptor family, it was suggested that OBP may
facilitate the transport of odorants rather than play a direct role in
olfactory signal transduction. However, this study should be now
reinvestigated in view of our current data since the major part of
radiolabelled bOBP used at the time was probably inactive. Binding parameters
have probably been largely underestimated in the absence of ligand
purification. Indeed, binding experiments reported in the present study
revealed that only a small fraction of pOBP is active upon iodination. With
the use of this purified, radiolabelled ligand, we could demonstrate a
high-affinity interaction between pOBP and OR. Furthermore, only one of the
two expressed receptors displayed recognition of this ligand, suggesting that
OBP may discriminate its OR partner. We are now investigating structurally
related lipocalins of different origins to get a better understanding of the
molecular basis supporting such an heterologous binding.
At the present time, the current understanding of olfactory perception is
based on three main observations: (i) pOBP selectively binds a panel of
odorants (Vincent et al.,
2000); (ii) OR activation by odorant is not dependent upon the
presence of OBP (Raming et al.,
1993; Krautwurst et
al., 1998; Malnic et
al., 1999; Wetzel et
al., 1999); and (iii) unliganded pOBP can selectively bind an
OR as shown herein. The formation of an OBP—OR—odorant complex, to
initiate olfactory response, is therefore questionable and remains to be
established. However, the current data do not rule out the hypothesis that OBP
may play a role in odorant removal to terminate the odorant response and
prevent receptor desensitization. Scheme
1 summarizes a putative multistep mechanism for odorant capture,
taking into account all the observations currently available. In the absence
of odorant, unoccupied OBP selectively binds to a relevant receptor with high
affinity and the OBP—OR binding stabilizes the receptor in a resting
state. When the odorant concentration is very low, virtually no odorant can by
itself reach the OR because its limited solubility prevents it from crossing
the mucus layer, and its uptake by OBP is favoured instead. The odorant-loaded
OBP may interact with an OR or an OBP—OR complex, depending on their
relative affinity. The odorant is then released and could activate the OR. As
the concentration increases, free odorant could also directly reach a specific
OR or displace an OBP—OR complex. In either case, odorant—OR
binding occurs and signal transduction can proceed. Under these conditions,
any odorant can be addressed to and taken up specifically to a relevant OR,
allowing discrimination and/or fine tuning of odorant recognition as a
function of both the nature and the amount of odorant. When the ligand
concentration is high, the scavenger role of OBP is dominant, thereby
preventing binding site saturation and protecting the OR from desensitization.
Elucidation of ligand specificity of OR 17-210 is now clearly needed to
validate these complex events. It is also necessary to identify if OR and OBP
subfamilies may respectively recognize structurally related odorants before it
can be concluded that OBP—OR recognition is of general physiological
relevance.
|
|
Acknowledgments |
|---|
We are very thankful to Professor Paolo Pelosi for constructive discussion and reading of this manuscript. We are also indebted to Professor Anny Cupo for fruitful discussion and support. We acknowledge the contribution of Dr Valérie Navarro, Dr Stéphane Martin and Dr Philippe Sarret (IPMC, CNRS UMR 6097, Sophia Antipolis, France) in designing the binding experiments. We are especially grateful to Stephane Canarelli (EPFL, Lausanne, Switzerland) for mass sprectrometry analysis. This work was supported in part by the Association pour l'Enseignement et le Développement de la Recherche en Région Provence-Alpes-Côte-d'Azur (ADER PACA).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ben-Arie, N., Lancet, D., Taylor, C., Khen, M., Walker, N., Ledbetter, D.H., Carrozzo, R., Patel, K., Sheer, D. and Lehrach, H. (1994) Olfactory receptor gene cluster on human chromosome 17: possible duplication of an ancestral receptor repertoire. Hum. Mol. Genet., 3,229 -235.
Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. and Amzel, L.M. (1996) The three-dimensional structure of bovine odorant-binding protein and its mechanism of odor recognition. Nat. Struct. Biol.,3 , 934-939.[Web of Science][Medline]
Blache, P., Gros, L., Salazar, G. and Bataille D. (1998) Cloning and tissue distribution of a new rat olfactory receptor-like (OL2). Biochem. Biophys. Res. Commun.,142 , 669-672.
Boudjelal, M., Sivaprasadarao, A. and Findlay, J. B. (1996) Membrane receptor for odour-binding proteins.J. Biochem. , 317,23 -27.
Breer, H. (1994) Odor recognition and second messenger signaling in olfactory receptor neurons. Semin. Cell Biol., 5,25 -32.[Medline]
Burova, T.V., Choiset, Y., Jankowski, C.K. and Haertle, T. (1999) Conformational stability and binding properties of porcine odorant binding protein. Biochemistry,38 , 15043-15051.[Medline]
Crowe, M.L., Perry, B.N. and Connerton, I.F. (1996) Olfactory receptor-encoding genes and pseudogenes are expressed in humans. Gene, 169,247 -249.[Web of Science][Medline]
Dal Monte, M., Andreini, I., Revoltella, R. and Pelosi, P. (1991) Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig.Comp. Biochem. Physiol. , 99,445 -451.
Dal Farra, C., Zsüger, N., Vincent, J.P. and Cupo, A. (2000) Binding of a pure monoiodoleptin analog to mouse tissues: a developmental study. Peptides,21 , 577-587.[Web of Science][Medline]
Drutel, G., Arrang, J.M., Diaz, J., Wisnewsky, C., Schwartz, K. and Schwartz, J.C. (1995) Cloning of OL1, a putative olfactory receptor and its expression in the developing rat heart. Receptors Channel, 3,33 -40.[Web of Science][Medline]
Ferrand, N., Pessah, M., Frayon, S., Marais, J. and Garel, J.M. (1999) Olfactory receptors, Golf alpha and adenylyl cyclase mRNA expressions in the rat heart during ontogenic development. J. Mol. Cell. Cardiol.,31 , 1137-1142.[Web of Science][Medline]
Flower, D.R. (1994) The lipocalin protein family: a role in cell regulation. FEBS Lett.,354 , 7-11.[Web of Science][Medline]
Flower, D.R. (1996) The lipocalin protein family: structure and function. Biochem. J.,318 , 1-14.
Gaudriault, G., Zsürger, N. and Vincent, J.P. (1994) Compared binding properties of 125I-labeled analogues of neurotensin and neuromedin N in rat and mouse brain.J. Neurochem. , 62,361 -368.[Web of Science][Medline]
Gimelbrant, A.A., Stoss, T.D., Landers, T.M. and McClintock, T.S. (1999) Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells. J. Neurochem., 72,2301 -2311.[Web of Science][Medline]
Gimelbrant, A.A, Haley, S.L. and McClintock, T.S.
(2001) Olfactory receptor. J. Biol.
Chem., 276,7285
-7290.
Ivic, L., Zhang, C., Zhang, X., Yoon, S.O. and Firenstein, S. (2002) Intracellular trafficking of a tagged and functional mammalian olfactory receptor. J. Neurobiol., 50,56 -58.[Web of Science][Medline]
Krautwurst, D., Yau, K.W. and Reed, R.R. (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell,95 , 917-926.[Web of Science][Medline]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature , 227,680 -685.[Medline]
Malnic, B., Hirono, J., Sato, T. and Buck L.B. (1999) Combinatorial receptor codes for odors.Cell , 96,713 -723.[Web of Science][Medline]
Marchalonis, J.J. (1969) An enzymic method for the trace iodination of immunoglobulins and other proteins.Biochem. J , 113,299 -305.[Web of Science][Medline]
Mombaerts, P. (1999) Molecular biology of odorant receptors in vertebrates. Annu. Rev. Neurosci.,22 , 487-509.[Web of Science][Medline]
Mombaerts, P. (2001) How smell develops. Nat. Neurosci Suppl., 4,1192 -1198.
Nef, S. and Nef, P. (1997)
Olfaction: transient expression of a putative odorant receptor in the
avian notochord. Proc. Natl Acad. Sci. USA,94
, 4766-4771.
Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima,
H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E.,
Yamamoto, M. and Miyano, M. (2000) Crystal
structure of rhodopsin: a G-protein-coupled receptor.Science
, 289,739
-745.
Paolini, S., Scaloni, A., Amoresano, A., Marchese, S.,
Napolitano, E. and Pelosi, P. (1998) Amino acid
sequence, post-translational modifications, binding and labelling of porcine
odorant-binding protein. Chem. Senses,23
, 689-698.
Parmentier, M., Libert, F., Schurmans, S., Schiffmann, S., Lefort, A., Eggerickx, D., Ledent, C., Mollereau, C., Gerard, C., Perret, J., Grootegoed, A. and Vassart, G. (1992) Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature, 355,453 -455.[Medline]
Pelosi, P., Baldaccini, N.E. and Pisanelli, A.M. (1982) Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine. Biochem. J.,201 , 245-248.[Web of Science][Medline]
Pelosi, P. (1994) Odorant-binding proteins. Crit. Rev. Biochem. Mol. Biol.,29 , 199-228.[Web of Science][Medline]
Pelosi, P. (1996) Perireceptor events in olfaction. J. Neurobiol., 30,3 -19.[Web of Science][Medline]
Perduca, M, Mancia F., Del Giorgio R. and Monaco H.L. (2001) Crystal structure of a truncated form of porcine odorant-binding protein. Proteins,42 , 201-209.[Web of Science][Medline]
Pevsner, J., Trifiletti, R.R., Strittmatter, S.M. and
Snyder, S.H. (1985) Isolation and characterization of
an olfactory receptor protein for odorant pyrazines. Proc. Natl
Acad. Sci. USA, 82,3050
-3054.
Pevsner, J., Hou, V., Snowman, A.M. and Snyder, S.H.
(1990) Odorant-binding protein: characterization of ligand
binding. J. Biol. Chem., 265,6118
-6125.
Raming, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstark, C. and Breer, H. (1993) Cloning and expression of odorant receptors. Nature,361 , 353-356.[Medline]
Raming, K., Konzelmann, S. and Breer, H. (1998) Identification of a novel G-protein coupled receptor expressed in distinct brain regions and a defined olfactory zone.Receptors Channels , 6,141 -151.[Web of Science][Medline]
Rouquier, S., Taviaux, S., Trask, B.J., Brand-Arpon, V., Van den Engh, G., Demaille, J. and Giorgi, D. (1998) Distribution of olfactory receptor genes in the human genome.Nat. Genet. , 18,243 -250.[Web of Science][Medline]
Sharon, D., Glusman, G., Pilpel, Y., Khen, M., Gruetzner, F., Haaf, T. and Lancet, D. (1999) Primate evolution of an olfactory receptor cluster: diversification by gene conversion and recent emergence of pseudogenes. Genomics,61 , 24-36.[Web of Science][Medline]
Spinelli, S., Ramoni, R., Grolli, S., Bonicel, J., Cambillau, C. and Tegoni, M. (1998) The structure of the monomeric porcine odorant-binding protein sheds light on the domain swapping mechanism. Biochemistry, 37,7913 -7918.[Medline]
Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. and Cambillau, C. (1996) Domain swapping creates a third putative combining site in bovine odorant-binding protein dimer.Nat. Struct. Biol. , 3,863 -867.[Web of Science][Medline]
Tegoni, M., Pelosi, P., Vincent, F., Spinelli, S., Campanacci, V., Grolli, S., Ramoni, R. and Cambillau, C. (2000) Mammalian odorant binding proteins. Biochim. Biophys. Acta, 1482,229 -240.[Medline]
Thomas, MB., Haines, S.L. and Akeson, R.A. (1996) Chemoreceptors expressed in taste, olfactory and male reproductive tissues. Gene, 178,1 -5.[Web of Science][Medline]
Vanderhaeghen, P., Schurmans, S., Vassart, G. and Parmentier, M. (1997) Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species.Genomics , 39,239 -246.[Web of Science][Medline]
Vincent, F., Spinelli, S., Ramoni, R., Grolli, S., Pelosi, P., Cambillau, C. and Tegoni, M. (2000) Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J. Mol. Biol.,300 , 127-239.[Web of Science][Medline]
Wetzel, C.H., Oles, M., Wellerdieck, C., Kuczkowiak, M.,
Gisselmann, G. and Hatt, H. (1999) Specificity and
sensitivity of a human olfactory receptor functionally expressed in human
embryonic kidney 293 cells and Xenopus laevis oocytes. J.
Neurosci., 19,7426
-7433.
Accepted July 16, 2002
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Hajjar, D. Perahia, H. Debat, C. Nespoulous, and C. H. Robert Odorant Binding and Conformational Dynamics in the Odorant-binding Protein J. Biol. Chem., October 6, 2006; 281(40): 29929 - 29937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Matarazzo, O. Clot-Faybesse, B. Marcet, G. Guiraudie-Capraz, B. Atanasova, G. Devauchelle, M. Cerutti, P. Etievant, and C. Ronin Functional Characterization of Two Human Olfactory Receptors Expressed in the Baculovirus Sf9 Insect Cell System Chem Senses, March 1, 2005; 30(3): 195 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sanz, C. Schlegel, J.-C. Pernollet, and L. Briand Comparison of Odorant Specificity of Two Human Olfactory Receptors from Different Phylogenetic Classes and Evidence for Antagonism Chem Senses, January 1, 2005; 30(1): 69 - 80. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||