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Pheromone binding proteins of the moth Mamestra brassicae: Specificity of ligand binding
Insect Biochem. Molec. Biol. Vol. 27, No. 3, pp. 213-221, 1997
Pergamon
PII: S0965-1748(96)00088-4
© 1997ElsevierScienceLtd All rightsreserved.Printedin GreatBritain 0965-1748/97 $17.00+ 0.00
Pheromone Binding Proteins of the Moth Mamestra brassicae: Specificity of Ligand Binding MARTINE MAIBI~CHE-COISNE,t FRANCK SOBRIO,~ THIERRY DELAUNAY,§ MARTINE L E T r E R E , t JACQUELINE DUBROCA,I" EMMANUELLE JACQUIN-JOLY,t PATRICIA NAGNAN-LE MEILLOUR*t Received 3 July 1996; revised and accepted 18 October 1996
Several isoforms of pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP) were previously characterized in the antennae of the cabbage armyworm Mamestra brassicae L. (Lepidoptera: Noctuldae). In further investigations, we used two-dimensional electrophoresis and Western-blotting with antibodies raised against the PBPs of the male: this method revealed more proteins with molecular weight and isoelectric points similar to those of OBPs and confirmed the high level of microdiversity suspected for this family of proteins. The binding of the tritiated m ~ o r pheromone compound, Zll-16:Ac, with male and female antennal extracts and purified PBPs from male antennae was studied. Only the two isoforms Mbra-1 and Mbra-l' (N-terminus: SKELI) bound the labelled pheromone, whereas no binding was observed with the Mbra-2 (N-terminus: SQEIM). In female antennal extracts, binding was shown between Zll.16:Ac and the proteins Mbra-1 and GOBP2. These results constitute an unambiguous demonstration of the binding specificity of a PBP to a pheromonal ligand, supporting the hypothesis of active participation of PBPs in odor discrimination, as a filter for odorants, prior to the receptor activation. © 1997 Elsevier Science Ltd. All rights reserved. Olfaction
Pheromone-binding protein
Lepidoptera Noctuidae Mamestra brassicae
INTRODUCTION
Ligand binding
tory receptors were unsuccessful in invertebrates. It is possible that the genes corresponding to olfactory recepEncoding of pheromonal odors in moths is probably the tors belong to distinct families sharing no homology best model of an olfactory system since detection is among them as in the case of the receptors of the devoted to only a few molecules in a very specialized vomeronasal and main olfactory systems in vertebrates system. Thus, the perception of a specific odor relies on (Dulac and Axel, 1995). Since the specificity of odor reca specialized olfactory receptor neuron (ORN) but the ognition by olfactory receptors cannot be tested, involvesuccession of events leading to the translation of this ment of another class of proteins, the odorant-binding message in term of specific behavior is unclear. The proteins (OBP) in perireceptor events, is widely studied. specificity in pheromone encoding was previously attriThese small soluble proteins are synthesised by accessory buted to specific olfactory receptors embedded in the cells and excreted into the sensillar lymph bathing the dendritic membranes of ORNs. These receptors were dendrites of ORNs (Kaissling, 1986; Steinbrecht et al., supposed to belong to the seven transmembrane domain 1992). In Lepidoptera, three groups were defined based receptor family characterized in the main olfactory epion amino acid sequence homologies. In one group the thelium of mammals (Buck and Axel, 1991). But pheromone-binding proteins (PBP) (Vogt and Riddiford, attempts to identify genes related to the family of olfac1981) constitute an heterogenous group in which the homology of PBPs among and within species is low (Vogt et al., 1989, 1991a; Krieger et al., 1991; Nagnan*Author for correspondence.E-mail: [email protected] tlNRA, Unit6de Phytopharmacieet des Mrdiateurs Chimiques,Route Le Meillour et al., 1996). Evidence of binding with de Saint-Cyr, F-78026, Versailles Cedex, France. pheromone compounds exists only for PBPs (Vogt and :~CEA, Service des Molrcules Marqures, Saclay, F-91191 Gif-surRiddiford, 1981; Vogt et al., 1989; Maida et al., 1993; Yvette, France. §INRA, Unit6 de Virologic et ImmunologieMolrculaires, F-78352 Du and Prestwich, 1995). Two other classes, distinguished by their N-terminal sequences, were found in Jouy-en-Josas Cedex, France. 213
214
MAIBI~CHE-COISNE MARTINE et al.
both sexes and were called general odorant-binding proteins (GOBP1 and GOBP2), based on sequence homology with PBPs, tissue specificity and pheromone binding (Breer et al., 1990; Vogt et al., 1991a, b). It has been shown that the distribution of OBPs in different kinds of sensilla can be related to the type of odorants detected, and associated with distinct classes of ORNs (Vogt et al., 1991a). Thus, the PBPs are located in the pheromonesensitive sensilla, the sensilla trichodea, while GOBPs are located in the sensilla basiconica, tuned to the detection of interspecific signals like volatiles from host-plants (Laue et al., 1994; Steinbrecht et al., 1995). Despite the evidence of pheromone binding by the PBP of Antherea polyphernus in early studies (Vogt and Riddiford, 1981), PBPs have been considered to perform nonspecific roles as solubilizers, carriers, and inactivators of pheromone molecules (Kaissling, 1986; Vogt, 1987; Van den Berg and Ziegelberger, 1991). However, the discovery of multiple forms of PBPs and GOBPs in an increasing number of moth species (Nagnan-Le Meillour et al., 1994, 1996; Maida et al., 1995; Steinbrecht et al., 1995; Prestwich et al., 1995; Krieger et al., 1996) suggests a high specificity for the odorants, especially between the PBPs and the pheromone compounds. However, demonstrating this specificity will require functional studies with large amounts of purified or recombinant protein as well as knowledge of sex pheromone encoding. In Lepidoptera, particularly in Noctuidae, the pheromonal blend emitted by females to attract conspecific males is generally composed of a restricted number of molecules derived from the fatty acid biosynthesis pathway. In the cabbage armyworm Mamestra brassicae, the pheromone is composed of a major component, Z l l 16:Ac (92%) and minor components, 16:Ac (7%), Z916:Ac (1%) (Farine et al., 1981), and ZII-16:Ald (0.1%)(Jacquin, 1992). Z11-16:Ac alone is able to attract conspecific males whereas Z9-16:Ac and the aldehyde synergize its effect (Farine et al., 1981; Attygalle et al., 1987; Subchev et al., 1985, 1987). The 16:Ac has no activity on behavior but is thought to be only an intermediate in the biosynthesis of the pheromones in the gland, since all identifications of the gland content were done by solvent extraction and not by head-space collection. Three receptor cell types were characterized in M. brassicae by single sensillum recordings (Renou and Lucas, 1994). Two cell types are located on the long lateral hairs, one type is tuned to the Z11-16:Ac, while the other is equally tuned to the Z9-14:Ac and Z11-16:OH, which are both known to decrease the males' attraction towards a pheromone source (antagonists). The third type of receptor cell is located in a different kind of sensilla trichodea, the short olfactory hairs, and is tuned to Z914:Ac (antagonist) and to ZI 1-16:Aid. No receptor cells were found to respond to Z9-16:Ac or to 16:Ac. OBPs from M. brassicae antennae were characterized and purified from both males and females by RP-HPLC
(Nagnan-Le Meillour et al., 1996). In male antennae, three isoforms of PBP migrate into two distinct bands after native-PAGE, and were called Mbra-1 (N-terminus: SKELI; band 2), Mbra-l' (SKELI; band 1) and Mbra-2 (SQEIM; band 1). In female antennae, only two PBPs were characterized, Mbra-1 (SKELI; band 2) and Mbra-2 (SQEIM; band 1). One GOBP2 was found in both sexes (TAEVM; band 3). In this paper, we report improvements in the resolution between different isoforms by two dimensional electrophoresis and immunodetection. So far, there are no binding data for pheromone ligands and purified PBP isoforms. In order to link biochemical and electrophysiological data in M. brassicae, we also investigated the binding of a tritiated analogue of the major compound of the pheromone of M. brassicae, the Z11-16:Ac, to purified Mbra-1, Mbra-l' and Mbra-2. MATERIALS AND METHODS
Insects
Animals were reared in Domaine du Magneraud (INRA, France) on semi-artificial diet (Poitout and Bues, 1974) at 24°C and 60% RH and sexed as pupae. The antennae of 3-day-old adults were excised and stored at - 8 0 ° C before protein extraction. Preparation o f samples
Two methods were used for the preparation of the crude tissue extracts: the antennae or other parts of the body were homogenized in a 1% trifluoroacetic acid solution, and then centrifuged (11 000 g, 15 min) with Zspin low binding filters (Gelman Sciences). Alternatively, the antennae were homogenized in a Tris/HCl buffer (Tris 20 raM, pH=7.4) using a Polytron TM homogenizer with a miniprobe and then centrifuged (10000 g, 30 min) (modified from Maida et al., 1993). Both extractions were done at 4°C. The resulting supernatants were evaporated in a speed-vac concentrator and stored at - 8 0 ° C until electrophoresis. Proteins were purified by RP-HPLC (Nagnan-Le Meillour et al., 1996): the purity of the fractions was checked by native-PAGE and Nterminal sequences were determined by gas-phase microsequencing (Jacques d'Alayer, Institut Pasteur, France). Preparation of antibodies
Ten-week-old female BALB/c mice were immunized in the rear footpads with 15 /zg of a mix of Mbra1/Mbra-l' or Mbra-2 from males (two mice per antigen) in Freund's complete adjuvant and were boosted 12 days later with the same amount of PBP in incomplete adjuvant. The popliteal lymph node cells were fused with the myeloma cells Sp2/0 in presence of PEG according to a standard protocol (Mirza et al., 1987). Selected hybridoma cells were cloned, expanded and produced in vitro. Non-related anti-plant and animal virus monoclonal antibodies were used as negative controls in ELISA
PHEROMONE-BINDINGPROTEINS OF MAMESTRA BRASSICAE screening. Supernatants of unfused Mbra-1- or Mbra-2stimulated lymph node cells were used as positive controis in the screening procedure. In the following experiments we used as primary antibodies these two polyclonal supernatants, called anti-Mbra-1 and anti-Mbra-2. The specificity of the antibodies was tested by immunoblotting on purified PBPs (described as follows).
Two-dimensional electrophoresis Two-dimensional electrophoresis was performed in a SE 220 tube gel adaptor kit for Hoefer SI mini-gel apparatus according to the manufacturer's directions (HSI). For isoelectric focusing, the anode was 0.085% phosphoric acid and the cathode 20 mM NaOH. The gels casted in tubes (5.6% T; 0.95% C, urea 9 M, nonidet NP 40 2%, ammonium persulfate 0.525 mM and Temed 0.325 mM) contained 2.0% of ampholytes of pH=4/6 (Ampholines, Pharmacia LKB Biotechnologies). The extracts were solubilized in sample buffer (O'Farrel et al., 1977). The gels were prefocused for 15 min at 250 V, then the migration was performed at room temperature for 4 h at 500 V. After migration, the gels were extracted from the tubes and equilibrated in 10% glycerol, 4.9 mM dithiothreitol, 2% SDS, bromophenol blue and 125 mM Tris/HCl, pH=6.8. For the second dimension, tube gel was sealed in a 1.5 mm thick SDS-PAGE peptide gel (Sch~igger and Von Jagow, 1987) with a plug of 1% agarose in the second-dimension sample buffer. The 16.5% T, 6% C separating gel was overlayed by a 4% T, 3% C stacking gel. Migration was initially conducted at 10 mA/gel for 15 min, followed by 4 h at 20 mA/gel at room temperature. Molecular weight standards consisted of a mixture of Pharmacia LMW (14.494 kDa) and Sigma PMW (2.5-16.9 kDa). Gels that were not blotted were fixed overnight in 50% methanol, 10% acetic acid, stained with 0.025 Serva Blue G in 10% acetic acid, and destained in 10% acetic acid.
Electroblotting and immunodetection Proteins were blotted onto PVDF (Immobilon® P, Millipore), as in Wiltfang et al. (1991). The buffer composition and procedure were described by Jacquin-Joly and Descoins (1996). The transfer was performed at 1.85 mA cm -2 of membrane for 1 h at room temperature. Non-specific sites on the membranes were blocked for 3x40 min in TBS (20 mM "Iris, 137 mM NaC1, pH=7.6) with 5% nonfat dry milk, briefly washed in TBS-0.1% Tween 20 and then incubated with an optimized concentration of primary antibodies (anti-Mbra-1 and/or anti-Mbra-2, dilution 1:300, overnight at 4°C) and secondary antibodies (horse radish peroxidase coupled anti-mouse IgG, dilution 1:6000, for 2 h at 37°C). After 3x30 min washing in TBS-0.1% Tween 20, the binding of the secondary antibodies was detected using the enhanced chemiluminescence kit (ECLTM, Amersham) and visualized after 3 min exposure on Hyperfilm MP (Amersham).
215
Synthesis of [11,12-3H]-(Z)-I 1-hexadecenyl acetate (1)
11-Hexadecynyl acetate (13.7 mg=0.05 mmol) in 2 ml ethyl acetate was partially reduced by 20 Ci (740 Gbq) of tritium gas introduced by means of a toepler pump. Lindlar catalyst (31.6 mg) poisoned by 10 ml of 5% quinoline in ethyl acetate was used as a catalyst. After 15 h of reaction at 1 atm, the catalyst was filtered off on a millex sr 0.5 /xm filter (Millipore) and labile tritium was removed by exchange with methanol and evaporation. The crude solution contained 4.4 Ci (162 Gbq) and about 65% of 1 (TLC: Merck RP18-F254s: acetonitrile: Rf=0.4). 1 was purified by HPLC (Zorbax ODS: 9.4x250 mm: acetonitrile: Rv=44 ml). The purity of the final product was checked by HPLC (Zorbax ODS: 4.6x250 mm: acetonitrile: Rt=5.34 mn: 98.7%) and TLC (Merck silice 60 F254 impregnated with silver nitrate: petroleum spirit 50/benzene 50: Rf=5.1: 99.8%). The co-migration of the non-labelled product and 1 was achieved in all cases. The specific radioactivity of 1.7 Tbq mmol-1 was determined by mass spectrometry (3H NMR: CDCl3/singlet; 5.35 ppm. Mass spectrometry: DCI]NH3: 304(M+18): 100; 300: 10; 302: 31; 305: 19; 306: 8). Binding study The binding of labelled Z11-16:Acetate to OBPs was measured by non-denaturing electrophoresis of incubated mixtures, with a protocol modified from Vogt et al. (1989). Lyophilized extracts of 50 antennae (male and female) and purified PBPs (3 /~g of each protein: Mbra1, Mbra-1 ', Mbra-2) were dissolved in 15 /xl of sample buffer (EDTA 1 mM, saccharose 20%, 10 mM Tris/HC1 pH=8), to a 15 /zM final concentration of each PBP. Tritiated Z11-16:Ac (2 /M, 2 /~Ci) in ethanol was added to each tube. The mixture was incubated for 30 min on ice before the samples were loaded on a native gel (16.8% separating gel, 4% stacking gel). Electrophoresis was performed at a constant voltage of 100 V for 1 h at room temperature. The gel was prepared for fluorography by fixation in 7% formaldehyde for 30 min following by a 1 h incubation in a 1 M salicylic acid solution. The gel was air dried overnight at room temperature between two sheets of cellophane paper and then exposed to film (Hyperfilm MP, Amersham) at -70°C for 1 week. As a control, the same extracts and proteins were separated under the same conditions at the same time but without incubation with the tritiated compound and the gel was stained with Coomassie blue.
RESULTS Antennal-specific proteins The electrophoretic pattern of antennal extracts under non-denaturing conditions showed five major bands in the acidic zone corresponding to highly mobile proteins with no qualitative differences between male and female (Nagnan-Le Meillour et al., 1996). Proteins extracted
MAIBI~CHE-COISNEMARTINE et al.
216
from other tissues migrated in the same positions as PBP bands from the antennae (Fig. l a). After electroblotting of the same samples onto PVDF membranes, immunodetection with a mixture of anti-Mbra-1 and anti-Mbra-2 antibodies (dilution 1:500) revealed strong labelling of bands 1 and 2 in antennal homogenates (Fig. l b) and limited labelling of bands from female legs in a region of lower mobility. Proteins of band 3 (GOBP2) did not cross-react with the antibodies.
Two-dimensional electrophoresis Since the SKELI sequence was observed as two distinct bands after native-PAGE in the male antennal homogenate (Mbra-1 and Mbra-1') and no difference was observed between male and female antennal homogenates after native-PAGE electrophoresis (Nagnan-Le Meillour et al., 1996), the proteins were analysed by twodimensional electrophoresis. After Coomassie staining (Fig. 2a), at least ten spots with a molecular weight between 15 and 16 kDa were observed in male homogenates, with two predominant spots at pls around 5.0 and 5.3. At least two different proteins with similar molecular weights were resolved in each spot (Fig. 2b). In the female extracts, there were less spots in the same pI range, with a major one at pI=5.0 (data not shown). After electroblotting and immunodetection, it was possible to distinguish the different PBPs. In male extracts, the mixture of the anti-Mbra-1 and anti-Mbra-2 antibodies stained strongly the two spots with pI=5.0 and pi=5.3
(Fig. 2c). The anti-Mbra-1 and anti-Mbra-2 antibodies alone stained the spots at pi=5.3 and pI=5.0 respectively (Fig. 2c). The same staining was observed in the female extracts. The isoelectric points of the PBPs of different species of Lepidoptera were calculated on the basis of amino acid composition, or measured (for A. polyphemus, Manduca sexta and Bombyx mori) within a narrow range, between 4.43 and 5.12 (Pelosi and Maida, 1995): our estimated pI are in agreement with these data. Two-dimensional electrophoresis provided a better separation of the proteins Mbra-1 and Mbra-2 in male and female homogenates than after native-PAGE, and showed some qualitative differences between the two sexes: fewer proteins were visualized in the female homogenates, and the spot containing the Mbra-! is less abundant in the female extracts. However, the proteins with the SKELI sequence (Mbra-1 and M b r a - l ' ) appear to have the same isoelectric point and their separation may need a higher resolution system, such as a Rotofor (BioRad; Prestwich, 1993), which generates a wider pH gradient.
Pheromone binding study After incubation of the 3H-Zll-16:Ac with the antennal homogenates from males and females or with the purified male PBPs, the protein-bound pheromone was analyzed by native-PAGE and fluorography (Fig. 3). In male antennal homogenates, the binding was localized only in the region of migration of the PBPs and no radio-
9 A~¢~
O)
~¢~"
~.~
/ II
(b)
PBPs
FIGURE 1. Non-denaturing polyacrylamidegel electrophoresis (native-PAGE, 16.8% acrylamide) of crude extracts from different tissues of male and female M. brassicae: head (1), thorax (1), antennae (50), legs (6), abdomen (3 segments) and 10 ~1 of female hemolymph. (a) Proteins were visualized by Coomassie blue staining (solution of 0.035% Blue-R in 12% trichloroacetic acid, 5% ethanol). (b) Western-blots of the same samples (10% of each sample) with primary anti-Mbra-1 and antiMbra-2 antibodies mixed (dilution 1:500, detection by chemiluminescence). Arrows indicate the positions of the PBPs and the GOBP2 in antennal extracts.
PHEROMONE-BINDING PROTEINS OF MAMESTRA BRASSICAE
(b) Detail of Coonmssie Staining
(a) Coomassie Blue Staining
pH
6,0
5,3 5,0
217
5,3 5,0
4,0
(c) Details of ECL Mixture of anti-Mbra-1 and anti-Mbra-2 antibodies
Anti-Mbra- 1 antibodies Anti-Mbra-2 antibodies
,1
5,35,0
5,3
5,0
FIGURE 2. Two-dimensional electrophoresis of antennal homogenates of male M. brassicae. The pH range of the first dimension gel is indicated under the gels and the position of some molecular weight standards (kDa) used in the second dimension gel (tricine SDS-PAGE) are indicated at the left of the gels. (a) Coomassie blue staining of male antennal extracts. (b) Detail of Coomassie staining, with the two principal spots in which several proteins were resolved. (c) Western-blots of male antennal extracts with details of ECL (use of the anti-Mbra-1 and anti-Mbra-2 antibodies mixed or separately).
(b) Autoradiography
(a) Coomassie Blue Staining
d
o,
S
FIGURE 3. Binding on gel between 3H-Z11-16:Ac and OBPs. Homogenates of M. brassicae antennae or purified Mbra-PBPs were incubated with the tritiated pheromone and electrophoresed. (a) Coomassie blue staining of crude antennal extracts and purified proteins, showing the positions of the PBPs and GOBP2 and their amounts. (b) Fluorography and autoradiography of the same samples showing the tritiated pheromone associated with the different proteins.
218
MAIBI~CHE-COISNE MARTINE et al.
activity was found associated with the third band corresponding to the GOBP2. In the female homogenates, the band 2 (Mbra-l: SKELI) and particularly the band 3 (GOBP2) were labelled. Only the male purified proteins Mbra- 1 and Mbra- 1 ', with the SKELI N-terminus, bound the pheromone in our experimental conditions and no binding was observed with the protein Mbra-2 (SQEIM). The sample preparation and the HPLC apparently did not alter the binding properties of the proteins. In addition, the binding between the proteins and the pheromonal compound appeared strong enough to remain stable during electrophoresis.
DISCUSSION The polyclonal antibodies raised against the PBPs of M. brassicae showed that these proteins are only present
in both male and female antennae and share this tissuespecificity with all other PBP studied in Lepidoptera (Vogt and Riddiford, 1981; Gy6rgyi et al., 1988; Vogt et al., 1989; Maida et al., 1993). No cross-reactivity was found with the GOBP2 (band 3) in the antennal extracts, which confirms the specificity of the antibodies tested. The faint staining observed only in the female legs is interesting and could be interpreted as a cross-reactivity with a protein of lower mobility which shares common epitopes with the PBPs. In Drosophila melanogaster, which has chemoreception sensory hairs in its legs like other Diptera, in situ hybridization between putative OBP clones and legs tissues has been reported (Pikielny et al., 1994), and recent data have indicated the existence of a putative odorant-binding protein in the antennae and legs of a Phasmid (Tuccini et al., 1996). Two-dimensional electrophoresis coupled with Western-blotting revealed additional proteins than previously detected, with a molecular weight (14-16 kDa) and an isoelectric point (4.5-6.0) alone compatible with PBP and GOBP. The use of the two antibodies raised against the PBP allowed us to separate clearly the Mbra- I' from the Mbra-2 which co-elute together in native-PAGE. In the males, the SKELI sequence was found in two distinct bands after native-PAGE (Mbra- 1 and Mbra- 1'), which could indicate either two different proteins with the same N-terminus or two forms of the same amino acid sequence with different arrangements of disulfide bridges, as assumed for the PBP of A. polyphemus by Ziegelberger (1996). After two-dimensional electrophoresis and MALDI-TOFMS (matrix assisted laser desorption/ionization-time of flight mass spectrometry, Blais et al., 1996), it is impossible to conclude because Mbra-I and Mbra-l' have close isoelectric points and close molecular weights (16 200 and 16 180 Da respectively). Separations using a Rotofor (Bio Rad) system are in progress (Maida et al., personal communication) to clarify this point. The study of binding between the tritiated major pheromonal compound and the antennal homogenates or pur-
ified PBPs of M. brassicae revealed important differences in the functional role of these proteins, as follows. Female extracts
The binding intensity is higher with the band 3 (GOBP2) than with the bands 1 and 2 (PBPs). In A. polyphemus, a binding between a protein belonging to the GOBP family and the pheromone component has been shown in the antennal extracts of female (Vogt and Riddiford, 1981) and male (Ziegelberger, 1996). However, in our species, the binding with the GOBP2 did not occur in male homogenate where PBP and GOBP are probably in competition. In this case, the affinity of the male PBPs for the Z l l - 1 6 : A c seems higher than those of the GOBP2. In the female extracts the PBPs are in smaller amounts than the GOBP2 and the Mbra-1' is absent. So, there are two hypotheses concerning the binding by the GOBP2 in the female extract. Either it could be the result of the small amount of Mbra-I present in the female, which explains the repartition of Z I 1-16:Ac between the two proteins. Or it could indicate that the female Mbra1 and the male Mbra-1 have different affinities for the Z11-16:Ac, so they could probably be two different isoforms of the same protein. Nevertheless, such a competition in the same sensilla in vivo appears uncertain: indeed, it has been demonstrated in several moth species that the GOBPs are only localized in sensilla basiconica and not co-expressed with the PBPs in sensilla trichodea (Laue et al., 1994; Steinbrecht et al., 1995). The binding between a GOBP2 and a sex pheromone is in agreement with the assumption of broad binding properties of this class of proteins (Vogt et al., 1991 a). The major point of interest is the binding observed between the Mbra-1 of the female homogenate and the pheromone component. It is the first time that such a binding has been observed in a female, but it has to be confirmed with the purified protein. In general, female moths have been viewed as anosmic towards their own pheromone. However, the presence of PBP in a female has been shown in some Lepidoptera species, such as Bombyx mori (Vogt et al., 1991a; Maida et al., 1993) and Manduca sexta (Gy6rgyi et al., 1988), suggesting that females may smell pheromone. Recently female perception of the conspecific pheromone was demonstrated in Spodoptera littoralis (Ljtinberg et al., 1993). Male extracts
The Mbra-1 and Mbra-1 ', which bind the major compound of the sex pheromone, are the most abundant PBPs present in the male antennal extract as established after HPLC and quantification (Bio-Rad protein Assay, data not shown). The hypothesis that each insect pheromone component could have a unique high-affinity PBP has been previously suggested (Du and Prestwich, 19951, supported by the presence of multiple PBPs in several moth species: two different PBPs have been previously identified in Antheraea pernyi and Lymantria dispar (Raming et al., 1990; Krieger et al., 1991; Vogt et al.,
PHEROMONE-BINDINGPROTEINS OF MAMESTRABRASSICAE 1989). In L. dispar, the binding between the two PBPs present in the sensillar extract with the two enantiomers of disparlure showed that one of the two proteins (Ldis2) has a stronger affinity than the other for both enantiomers, and it has been suggested that the other one (Ldis1) could specifically bind another component of the pheromone (Vogt et al., 1989) still unknown. On the other hand, the ligand specificity of the two recombinant PBPs from A. pernyi (Aper-1, Aper-2) and the recombinant PBP of A. polyphemus (Apol-3) for two pheromonal components were previously determined (Du and Prestwich, 1995): the two recombinant PBPs of the same species showed opposite binding specificity for two different ligands, whereas Aper-1 and Apol-3 shared high sequence homology and exhibit both a high affinity to the same pheromone compound. These authors suggest that the ligand specificity is probably encoded in the primary structure of the proteins, which have evolved to bind unique ligands. Our results show that two native purified PBPs of the same species exhibit opposite affinities for its major pheromone compound, and are consistent with the data obtained with recombinant proteins. These results of binding lead to several hypotheses to account for the functional role of the PBPs in Mamestra brassicae, according to the data known about the composition of the pheromone and the sensory encoding in this species, in relationship with the different morphological types of sensilla characterized (Fig. 4). We can propose that in M. brassicae male antennae, the Mbra-1 and Mbra-1' occur in the long sensory hairs, where they bind the Zll-16:Ac, which is known to stimulate one of the two receptor cells (ORN A). The Mbra-2 could be devoted to the binding of other pheromone components (intra- or interspecific ones). This protein could be either coexpressed in the long sensilla trichodea (where the second ORN B is equally tuned to Z9-14:Ac and to Z1 lL o n g sensilla trichodea
Z9-14:Ae Zll-16:OH
1-16:Ac~
ra-1
i
'
Mbra-1 ? Mbra-2 ?
219
16:OH, two compounds that decrease the flight response of males to pheromone sources), or only expressed in the short sensilla trichodea (which ORN respond to the Z914:Ac, antagonist and the Zll-16:AId, synergist at 0.1%). Immunohistological studies with the monoclonal antibodies raised against the different PBPs of M. brassicae are in progress to clarify these hypothesis. Moreover, the protein Mbra-2 shares a high N-terminus homology (88%) with the PBP of another noctuid moth, Heliothis virescens, whose major pheromonal compound is the Z11-16:Aid (Krieger et al., 1993): this component also is a pheromonal compound in M. brassicae and could be a putative ligand for Mbra-2. In addition, the PBP of two sibling species, Spodoptera descoinsi and S. latifascia (Nagnan-Le Meillour et al., in prep.) crossreacted with the anti-Mbra-2, and their N-terminus was determined to belong to the type Mbra-2 (SQELMVKM). These two species use the Z9-14:Ac as a component of the pheromone, and it could be a ligand for the Mbra-2 too. It would be of great interest to determine what kind of PBP is present in the species using Z11-16:Ac in their pheromone. Nevertheless, in closely related species, a structure-function relationship for the PBPs can be established. In Noctuidae, the detection of heterospecific pheromone compounds was clearly established (Lucas and Renou, 1989). The presence of ORN tuned to heterospecific pheromone compounds could be correlated at the level of perireceptor events to the expression of specific PBPs, which might be selected to recognize molecules that do not belong to the species. This adaptive mechanism could participate in the premating isolation between sympatric species, which are in competition for resources.
Short sensilla trichodea
ORN C Z9-14:Ae e Z11-16:Ald
ORN D .9
Mbra-1 ? Mbra-2 ?
I j l i Q I o° H'llUUlol,glml..imlnlUNiNglll''ll
FIGURE4. Hypotheticalmodel for the pheromonalencoding in Mamestra brassicae. ORN, olfactoryreceptor neuron; +, activator; -, inhibitor; Zll-16:Ac=ll-cis-hexadecenyl acetate; Zll-16:Ald=ll-cis-hexadecenyl aldehyde; Z9-14:Ac=9-cistetradecenyl acetate; ZI 1-16:OH=I1-cis-hexadecenol. It is possible to associate ORN A with the PBPs Mbra-I and Mbra-l' and with the majorcomponentof the pheromone,the Z11-16:Ac. Other associationsare still hypothetical.
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REFERENCES Attygalle A. B., Herrig M., Vostrowsky O. and Bestmann H. J. (1987) Technique for injecting intact glands for analysis of sex pheromones of Lepidoptera by capillary gas chromatography: Reinvestigation of pheromone complex of Mamestra brassicae. J. Chem. Ecol. 13, 1299-1311. Blais J. C., Nagnan-Le Meillour P., Bolbach G. and Tabet J. C. (1996) MALDI-TOFMS identification of odorant-binding proteins electroblotted onto poly(vinylidene difluoride) membranes. R. C. Mass Spectrom. 10, 1-4. Breer H., Krieger J. and Raming K. (1990) A novel class of bindingprotein in the antennae of the silkmoth Antheraea pernyi. Insect Biochem. 20, 735-740. Buck L. and Axel R. (1991) A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65, 175-187. Du G. and Prestwich G. D. (1995) Protein structure encodes the ligand binding specificity in pheromone-binding proteins. Biochemistry 34, 8726-8732. Dulac C. and Axel R. (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83, 195-206. Farine J. P., Fr6rot B. and Isart J. (1981) Facteurs d'isolement chimique dans la s6cr6tion ph6romonale de deux noctuelles Hadeninae: Mamestra brassicae (L.) et Pseudatelia unipuncta (Haw.). C. R. Acad. Sci. Paris (Sdrie I11) 292, 101-104. GyOrgyi T. K., Roby-Shemkovitz A. J. and Lerner M. R. (1988) Characterization and cDNA cloning of the pheromone-binding protein from the tabacco hornworm, Manduca sexta: a tissue-specific developmentally regulated protein. Proc. Nat. Acad. Sci. U.S.A. 85, 9851-9855. Jacquin E. (1992) REgulation neuroendocrine de la production des ph6romones sexuelles femelles chez deux noctuelles, Mamestra brassicae L. et Heliothis zea Boddie (L~pidopt6res), 131 pp. Thbse de I'INA-PG, Paris, France. Jacquin-Joly E. and Descoins C. (1996) Identification of PBAN-like peptides in the brain subesophageal ganglion complex of Lepidoptera using Western-blotting. Insect Biochem. Molec. Biol. 26, 209-216. Kaissling K. E. (1986) Chemo-electrical transduction in insect olfactory receptors. Ann. Rev. Neurosci. 9, 121-145. Krieger J., Raming K. and Breer H. (1991) Cloning of genomic and complementary DNA encoding insect PBP: evidence for microdiversity. Biochem. Biophys. Acta 1088, 277-284. Krieger J., Ganl31e H., Raining K. and Breer H. (1993) Odorant-binding protein of Heliothis virescens. Insect Biochem. Molec. Biol. 23, 449-456. Krieger J., Von Nickisch-Rosenegk E., Mameli M., Pelosi P. and Breer H. (1996) Binding proteins from the antennae of Bombyx mori. Insect Biochem. Molec. Biol. 26, 297-307. Laue M., Steinbrecht R. A. and Ziegelberger G. (1994) lmmunocytochemical localization of general odorant-binding proteins in olfactory sensilla of the silkmoth Antheraea polvphemus. Naturwissenschafien 81, 178-181. Lucas P. and Renou R. (1989) Responses to pheromone compounds in Mamestra suasa (Lepidoptera: Noctuidae) olfactory neurons. J. Insect Physiol. 35, 837-845. Ljiinberg H., Anderson P. and Hanson B. S. (1993) Physiology and morphology of pheromone-specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera: Noctuidae). J. Insect Physiol. 39, 253-260. Maida R., Steinbrecht A., Ziegelberger G. and Pelosi P. (1993) The pheromone binding protein of Bombyx mori: Purification, characterization and immunocytochemical localization. Insect Biochem. Molec. Biol. 23, 243-253. Maida R., Laue M., Steinbrecht R. A. and Ziegelberger G. (1995) Biochemical and immunocytochemical characterization of odorantbinding proteins in moths. In GOttingen Neurobiology Conference 1995 (Edited by N. Alsner and R. Menzel), Vol. II, p. 373. Thieme, Stuttgart, New York.
Mirza I. H., Wilkin T. J., Cantarini M. and Moore K. (1987) A comparison of spleen and lymph node cells as fusion partners for the raising of monoclonal antibodies after different route of immunization. J. Immun. Meth. 105, 235-241. Nagnan-Le Meillour P., Huet J. C., Malb~che M., Pernollet J. C., and Descoins C. (1994) Microdiversity of odorant-binding proteins in insects: the first step of odor discrimination. In Chemical Communication in Vertebrates and Invertebrates: Nature, Neuroregulation and Molecular Receptors o f Pheromones. Abstracts of the Jacques Monod Conference, 75 (Edited by J. L. CI6ment and D. Morgan). Nagnan-Le Meillour P., Huet J. C., Maib~che M., Pernollet J. C. and Descoins C. (1996) Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of Mamestra brassicae (Noctuidae). Insect Biochem. Molec. Biol. 26, 59~57. Nagnan-Le Meillour P., Blais J. C., Jacquin-Joly E., Mffl"o6che M., Tabet J. C. and Descoins C. (in preparation) Comparative studies of odorant-binding proteins in various insect orders and characterization of new OBPs in Noctuid species. O'Farrel P. Z., Goodman H. M. and O'Farrel P. H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142. Pelosi P. and Maida R. (1995) Odorant-binding proteins in insects. Comp. Biochem. Physiol. l l l B , 503-514. Pikielny C. W., Hasan G., Rouyer F. and Rosbach M. (1994) Members of a family of Drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs. Neuron 12, 35-49. Poitout S. and Bues R. (1974) Elevage de 28 esp6ces de Noctuidae et de 2 espbces d'Arctiidae sur milieu artificiel simplifi6. Particularit6s selon les espbces. Ann. Zool. Ecol. Anim. 6, 431-441. Prestwich G. D. (1993) Bacterial expression and photoaffinity labeling of a pheromone binding protein. Protein Sci. 2, 420-428. Prestwich G. D., Du G. and LaForest S. (1995) How is pheromone specificity encoded in proteins'? Chem. Senses 20, 461-469. Raining K., Krieger J. and Breer H. (1990) Primary structure of a pheromone-binding protein from Antheraea pernyi: homologies with other ligand-carrying proteins. Comp. Physiol. B. 160, 503 509. Renou M. and Lucas P. (1994) Sex pheromone reception in Mamestra brassicae (Lepidoptera): responses of olfactory receptor neurones to minor components of the pheromone blend. J. Insect Physiol. 40, 75-85. Sch~igger H. and Von Jagow G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis lor the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368379. Steinbrecht R. A., Ozaki M. and Ziegelberger G. (1992) Immunocytochemical localization of pheromone-binding protein in moth antennae. Cell Tissue Res. 270, 287-302. Steinbrecht R. A., Laue M. and Ziegelberger G. (1995) Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx. Cell Tissue Res. 282, 203-217. Subchev M. A., Stanimirova L. S. and Stoilov I. L. (1985) Effect ~1 cis-1 I-hexadecenol and its derivatives on the pheromonal activity of cis-l I-hexadecenyl acetate to males of three noctuid species (Lepidoptera: Noctuidae) in field. Ecology 17, 56-61. Subchev M. A., Stanimirova L. S. and Milkova T. S. (1987) The effect of compounds related to cis-11-hexadecenyl acetate on its attractiveness to the males of Mamestra brassicae L. (Lepidoptera, Noctuidae) and some other noctuid species. Folia Biol. 35, 143
150. Tuccini A., Maida R., Rovero P., Mazza M. and Pelosi P. (1996) Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea). Insect Biochem. Molec. Biol. 26, 19-24. Van Den Berg M. J. and Ziegelberger G. (1991) On the function of the pheromone-binding protein in the olfactory hairs of Antherea polyphemus. J. Insect Physiol. 37, 79-85.
PHEROMONE=BINDING PROTEINS OF MAMESTRA BRASSICAE Vogt R. G. and Riddiford L. M. (1981) Pheromone binding and inactivation by moth antennae. Nature 293, 161-163. Vogt R. G. (1987) The molecular basis of pheromone reception: its influence on behavior. In Pheromone Biochemistry (Edited by Prestwich G. D. and Blomquist G. J.), pp. 385--431. Academic Press, Orlando, FL. Vogt R. G., Kt~hne A. C., Dubnau J. T. and Prestwich G. D. (1989) Expression of pheromone-binding proteins during antennal development in the gypsy moth Lymantria dispar. J. Neurosci. 9, 3332-3346. Vogt R. G., Prestwich G. D. and Lerner M. R. (1991a) Odorant-binding protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiol. 22, 74-84. Vogt R. G., Rybczynski R. and Lerner M. R. (1991b) Molecular cloning and sequencing of general odorant-binding proteins GOBPI and GOBP2 from the tabacco hawk moth Manduca sexta: comparison with other insect OBPs and their signal peptides. J. Neurosci. 11, 2972-2984.
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Wiltfang J., Arold N. and Neuhoff V. (1991 ) A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100 0001000, and their detection with picomolar sensitivity. Electrophoresis 12, 352-366. Ziegelberger G. (1996) Redox-shift of the pheromone binding protein in the silkmoth Antheraea polyphemus. Fur. J. Biochem. 232~ 706-71 I.
Acknowledgements--The authors are grateful to Mafie-Th6r~se Bethenod for technical assistance. P.N.L.M. is indebted to Glenn Prestwich for constant encouragement. This work is dedicated to Charles Descoins. Research supported by INRA. We thank Dick Vogt, Michael Burnet, Paolo Pelosi and an anonymous reviewer for helpful comments on the manuscript.
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Pheromone Binding Proteins of the Moth Mamestra brassicae: Specificity of Ligand Binding MARTINE MAIBI~CHE-COISNE,t FRANCK SOBRIO,~ THIERRY DELAUNAY,§ MARTINE L E T r E R E , t JACQUELINE DUBROCA,I" EMMANUELLE JACQUIN-JOLY,t PATRICIA NAGNAN-LE MEILLOUR*t Received 3 July 1996; revised and accepted 18 October 1996
Several isoforms of pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP) were previously characterized in the antennae of the cabbage armyworm Mamestra brassicae L. (Lepidoptera: Noctuldae). In further investigations, we used two-dimensional electrophoresis and Western-blotting with antibodies raised against the PBPs of the male: this method revealed more proteins with molecular weight and isoelectric points similar to those of OBPs and confirmed the high level of microdiversity suspected for this family of proteins. The binding of the tritiated m ~ o r pheromone compound, Zll-16:Ac, with male and female antennal extracts and purified PBPs from male antennae was studied. Only the two isoforms Mbra-1 and Mbra-l' (N-terminus: SKELI) bound the labelled pheromone, whereas no binding was observed with the Mbra-2 (N-terminus: SQEIM). In female antennal extracts, binding was shown between Zll.16:Ac and the proteins Mbra-1 and GOBP2. These results constitute an unambiguous demonstration of the binding specificity of a PBP to a pheromonal ligand, supporting the hypothesis of active participation of PBPs in odor discrimination, as a filter for odorants, prior to the receptor activation. © 1997 Elsevier Science Ltd. All rights reserved. Olfaction
Pheromone-binding protein
Lepidoptera Noctuidae Mamestra brassicae
INTRODUCTION
Ligand binding
tory receptors were unsuccessful in invertebrates. It is possible that the genes corresponding to olfactory recepEncoding of pheromonal odors in moths is probably the tors belong to distinct families sharing no homology best model of an olfactory system since detection is among them as in the case of the receptors of the devoted to only a few molecules in a very specialized vomeronasal and main olfactory systems in vertebrates system. Thus, the perception of a specific odor relies on (Dulac and Axel, 1995). Since the specificity of odor reca specialized olfactory receptor neuron (ORN) but the ognition by olfactory receptors cannot be tested, involvesuccession of events leading to the translation of this ment of another class of proteins, the odorant-binding message in term of specific behavior is unclear. The proteins (OBP) in perireceptor events, is widely studied. specificity in pheromone encoding was previously attriThese small soluble proteins are synthesised by accessory buted to specific olfactory receptors embedded in the cells and excreted into the sensillar lymph bathing the dendritic membranes of ORNs. These receptors were dendrites of ORNs (Kaissling, 1986; Steinbrecht et al., supposed to belong to the seven transmembrane domain 1992). In Lepidoptera, three groups were defined based receptor family characterized in the main olfactory epion amino acid sequence homologies. In one group the thelium of mammals (Buck and Axel, 1991). But pheromone-binding proteins (PBP) (Vogt and Riddiford, attempts to identify genes related to the family of olfac1981) constitute an heterogenous group in which the homology of PBPs among and within species is low (Vogt et al., 1989, 1991a; Krieger et al., 1991; Nagnan*Author for correspondence.E-mail: [email protected] tlNRA, Unit6de Phytopharmacieet des Mrdiateurs Chimiques,Route Le Meillour et al., 1996). Evidence of binding with de Saint-Cyr, F-78026, Versailles Cedex, France. pheromone compounds exists only for PBPs (Vogt and :~CEA, Service des Molrcules Marqures, Saclay, F-91191 Gif-surRiddiford, 1981; Vogt et al., 1989; Maida et al., 1993; Yvette, France. §INRA, Unit6 de Virologic et ImmunologieMolrculaires, F-78352 Du and Prestwich, 1995). Two other classes, distinguished by their N-terminal sequences, were found in Jouy-en-Josas Cedex, France. 213
214
MAIBI~CHE-COISNE MARTINE et al.
both sexes and were called general odorant-binding proteins (GOBP1 and GOBP2), based on sequence homology with PBPs, tissue specificity and pheromone binding (Breer et al., 1990; Vogt et al., 1991a, b). It has been shown that the distribution of OBPs in different kinds of sensilla can be related to the type of odorants detected, and associated with distinct classes of ORNs (Vogt et al., 1991a). Thus, the PBPs are located in the pheromonesensitive sensilla, the sensilla trichodea, while GOBPs are located in the sensilla basiconica, tuned to the detection of interspecific signals like volatiles from host-plants (Laue et al., 1994; Steinbrecht et al., 1995). Despite the evidence of pheromone binding by the PBP of Antherea polyphernus in early studies (Vogt and Riddiford, 1981), PBPs have been considered to perform nonspecific roles as solubilizers, carriers, and inactivators of pheromone molecules (Kaissling, 1986; Vogt, 1987; Van den Berg and Ziegelberger, 1991). However, the discovery of multiple forms of PBPs and GOBPs in an increasing number of moth species (Nagnan-Le Meillour et al., 1994, 1996; Maida et al., 1995; Steinbrecht et al., 1995; Prestwich et al., 1995; Krieger et al., 1996) suggests a high specificity for the odorants, especially between the PBPs and the pheromone compounds. However, demonstrating this specificity will require functional studies with large amounts of purified or recombinant protein as well as knowledge of sex pheromone encoding. In Lepidoptera, particularly in Noctuidae, the pheromonal blend emitted by females to attract conspecific males is generally composed of a restricted number of molecules derived from the fatty acid biosynthesis pathway. In the cabbage armyworm Mamestra brassicae, the pheromone is composed of a major component, Z l l 16:Ac (92%) and minor components, 16:Ac (7%), Z916:Ac (1%) (Farine et al., 1981), and ZII-16:Ald (0.1%)(Jacquin, 1992). Z11-16:Ac alone is able to attract conspecific males whereas Z9-16:Ac and the aldehyde synergize its effect (Farine et al., 1981; Attygalle et al., 1987; Subchev et al., 1985, 1987). The 16:Ac has no activity on behavior but is thought to be only an intermediate in the biosynthesis of the pheromones in the gland, since all identifications of the gland content were done by solvent extraction and not by head-space collection. Three receptor cell types were characterized in M. brassicae by single sensillum recordings (Renou and Lucas, 1994). Two cell types are located on the long lateral hairs, one type is tuned to the Z11-16:Ac, while the other is equally tuned to the Z9-14:Ac and Z11-16:OH, which are both known to decrease the males' attraction towards a pheromone source (antagonists). The third type of receptor cell is located in a different kind of sensilla trichodea, the short olfactory hairs, and is tuned to Z914:Ac (antagonist) and to ZI 1-16:Aid. No receptor cells were found to respond to Z9-16:Ac or to 16:Ac. OBPs from M. brassicae antennae were characterized and purified from both males and females by RP-HPLC
(Nagnan-Le Meillour et al., 1996). In male antennae, three isoforms of PBP migrate into two distinct bands after native-PAGE, and were called Mbra-1 (N-terminus: SKELI; band 2), Mbra-l' (SKELI; band 1) and Mbra-2 (SQEIM; band 1). In female antennae, only two PBPs were characterized, Mbra-1 (SKELI; band 2) and Mbra-2 (SQEIM; band 1). One GOBP2 was found in both sexes (TAEVM; band 3). In this paper, we report improvements in the resolution between different isoforms by two dimensional electrophoresis and immunodetection. So far, there are no binding data for pheromone ligands and purified PBP isoforms. In order to link biochemical and electrophysiological data in M. brassicae, we also investigated the binding of a tritiated analogue of the major compound of the pheromone of M. brassicae, the Z11-16:Ac, to purified Mbra-1, Mbra-l' and Mbra-2. MATERIALS AND METHODS
Insects
Animals were reared in Domaine du Magneraud (INRA, France) on semi-artificial diet (Poitout and Bues, 1974) at 24°C and 60% RH and sexed as pupae. The antennae of 3-day-old adults were excised and stored at - 8 0 ° C before protein extraction. Preparation o f samples
Two methods were used for the preparation of the crude tissue extracts: the antennae or other parts of the body were homogenized in a 1% trifluoroacetic acid solution, and then centrifuged (11 000 g, 15 min) with Zspin low binding filters (Gelman Sciences). Alternatively, the antennae were homogenized in a Tris/HCl buffer (Tris 20 raM, pH=7.4) using a Polytron TM homogenizer with a miniprobe and then centrifuged (10000 g, 30 min) (modified from Maida et al., 1993). Both extractions were done at 4°C. The resulting supernatants were evaporated in a speed-vac concentrator and stored at - 8 0 ° C until electrophoresis. Proteins were purified by RP-HPLC (Nagnan-Le Meillour et al., 1996): the purity of the fractions was checked by native-PAGE and Nterminal sequences were determined by gas-phase microsequencing (Jacques d'Alayer, Institut Pasteur, France). Preparation of antibodies
Ten-week-old female BALB/c mice were immunized in the rear footpads with 15 /zg of a mix of Mbra1/Mbra-l' or Mbra-2 from males (two mice per antigen) in Freund's complete adjuvant and were boosted 12 days later with the same amount of PBP in incomplete adjuvant. The popliteal lymph node cells were fused with the myeloma cells Sp2/0 in presence of PEG according to a standard protocol (Mirza et al., 1987). Selected hybridoma cells were cloned, expanded and produced in vitro. Non-related anti-plant and animal virus monoclonal antibodies were used as negative controls in ELISA
PHEROMONE-BINDINGPROTEINS OF MAMESTRA BRASSICAE screening. Supernatants of unfused Mbra-1- or Mbra-2stimulated lymph node cells were used as positive controis in the screening procedure. In the following experiments we used as primary antibodies these two polyclonal supernatants, called anti-Mbra-1 and anti-Mbra-2. The specificity of the antibodies was tested by immunoblotting on purified PBPs (described as follows).
Two-dimensional electrophoresis Two-dimensional electrophoresis was performed in a SE 220 tube gel adaptor kit for Hoefer SI mini-gel apparatus according to the manufacturer's directions (HSI). For isoelectric focusing, the anode was 0.085% phosphoric acid and the cathode 20 mM NaOH. The gels casted in tubes (5.6% T; 0.95% C, urea 9 M, nonidet NP 40 2%, ammonium persulfate 0.525 mM and Temed 0.325 mM) contained 2.0% of ampholytes of pH=4/6 (Ampholines, Pharmacia LKB Biotechnologies). The extracts were solubilized in sample buffer (O'Farrel et al., 1977). The gels were prefocused for 15 min at 250 V, then the migration was performed at room temperature for 4 h at 500 V. After migration, the gels were extracted from the tubes and equilibrated in 10% glycerol, 4.9 mM dithiothreitol, 2% SDS, bromophenol blue and 125 mM Tris/HCl, pH=6.8. For the second dimension, tube gel was sealed in a 1.5 mm thick SDS-PAGE peptide gel (Sch~igger and Von Jagow, 1987) with a plug of 1% agarose in the second-dimension sample buffer. The 16.5% T, 6% C separating gel was overlayed by a 4% T, 3% C stacking gel. Migration was initially conducted at 10 mA/gel for 15 min, followed by 4 h at 20 mA/gel at room temperature. Molecular weight standards consisted of a mixture of Pharmacia LMW (14.494 kDa) and Sigma PMW (2.5-16.9 kDa). Gels that were not blotted were fixed overnight in 50% methanol, 10% acetic acid, stained with 0.025 Serva Blue G in 10% acetic acid, and destained in 10% acetic acid.
Electroblotting and immunodetection Proteins were blotted onto PVDF (Immobilon® P, Millipore), as in Wiltfang et al. (1991). The buffer composition and procedure were described by Jacquin-Joly and Descoins (1996). The transfer was performed at 1.85 mA cm -2 of membrane for 1 h at room temperature. Non-specific sites on the membranes were blocked for 3x40 min in TBS (20 mM "Iris, 137 mM NaC1, pH=7.6) with 5% nonfat dry milk, briefly washed in TBS-0.1% Tween 20 and then incubated with an optimized concentration of primary antibodies (anti-Mbra-1 and/or anti-Mbra-2, dilution 1:300, overnight at 4°C) and secondary antibodies (horse radish peroxidase coupled anti-mouse IgG, dilution 1:6000, for 2 h at 37°C). After 3x30 min washing in TBS-0.1% Tween 20, the binding of the secondary antibodies was detected using the enhanced chemiluminescence kit (ECLTM, Amersham) and visualized after 3 min exposure on Hyperfilm MP (Amersham).
215
Synthesis of [11,12-3H]-(Z)-I 1-hexadecenyl acetate (1)
11-Hexadecynyl acetate (13.7 mg=0.05 mmol) in 2 ml ethyl acetate was partially reduced by 20 Ci (740 Gbq) of tritium gas introduced by means of a toepler pump. Lindlar catalyst (31.6 mg) poisoned by 10 ml of 5% quinoline in ethyl acetate was used as a catalyst. After 15 h of reaction at 1 atm, the catalyst was filtered off on a millex sr 0.5 /xm filter (Millipore) and labile tritium was removed by exchange with methanol and evaporation. The crude solution contained 4.4 Ci (162 Gbq) and about 65% of 1 (TLC: Merck RP18-F254s: acetonitrile: Rf=0.4). 1 was purified by HPLC (Zorbax ODS: 9.4x250 mm: acetonitrile: Rv=44 ml). The purity of the final product was checked by HPLC (Zorbax ODS: 4.6x250 mm: acetonitrile: Rt=5.34 mn: 98.7%) and TLC (Merck silice 60 F254 impregnated with silver nitrate: petroleum spirit 50/benzene 50: Rf=5.1: 99.8%). The co-migration of the non-labelled product and 1 was achieved in all cases. The specific radioactivity of 1.7 Tbq mmol-1 was determined by mass spectrometry (3H NMR: CDCl3/singlet; 5.35 ppm. Mass spectrometry: DCI]NH3: 304(M+18): 100; 300: 10; 302: 31; 305: 19; 306: 8). Binding study The binding of labelled Z11-16:Acetate to OBPs was measured by non-denaturing electrophoresis of incubated mixtures, with a protocol modified from Vogt et al. (1989). Lyophilized extracts of 50 antennae (male and female) and purified PBPs (3 /~g of each protein: Mbra1, Mbra-1 ', Mbra-2) were dissolved in 15 /xl of sample buffer (EDTA 1 mM, saccharose 20%, 10 mM Tris/HC1 pH=8), to a 15 /zM final concentration of each PBP. Tritiated Z11-16:Ac (2 /M, 2 /~Ci) in ethanol was added to each tube. The mixture was incubated for 30 min on ice before the samples were loaded on a native gel (16.8% separating gel, 4% stacking gel). Electrophoresis was performed at a constant voltage of 100 V for 1 h at room temperature. The gel was prepared for fluorography by fixation in 7% formaldehyde for 30 min following by a 1 h incubation in a 1 M salicylic acid solution. The gel was air dried overnight at room temperature between two sheets of cellophane paper and then exposed to film (Hyperfilm MP, Amersham) at -70°C for 1 week. As a control, the same extracts and proteins were separated under the same conditions at the same time but without incubation with the tritiated compound and the gel was stained with Coomassie blue.
RESULTS Antennal-specific proteins The electrophoretic pattern of antennal extracts under non-denaturing conditions showed five major bands in the acidic zone corresponding to highly mobile proteins with no qualitative differences between male and female (Nagnan-Le Meillour et al., 1996). Proteins extracted
MAIBI~CHE-COISNEMARTINE et al.
216
from other tissues migrated in the same positions as PBP bands from the antennae (Fig. l a). After electroblotting of the same samples onto PVDF membranes, immunodetection with a mixture of anti-Mbra-1 and anti-Mbra-2 antibodies (dilution 1:500) revealed strong labelling of bands 1 and 2 in antennal homogenates (Fig. l b) and limited labelling of bands from female legs in a region of lower mobility. Proteins of band 3 (GOBP2) did not cross-react with the antibodies.
Two-dimensional electrophoresis Since the SKELI sequence was observed as two distinct bands after native-PAGE in the male antennal homogenate (Mbra-1 and Mbra-1') and no difference was observed between male and female antennal homogenates after native-PAGE electrophoresis (Nagnan-Le Meillour et al., 1996), the proteins were analysed by twodimensional electrophoresis. After Coomassie staining (Fig. 2a), at least ten spots with a molecular weight between 15 and 16 kDa were observed in male homogenates, with two predominant spots at pls around 5.0 and 5.3. At least two different proteins with similar molecular weights were resolved in each spot (Fig. 2b). In the female extracts, there were less spots in the same pI range, with a major one at pI=5.0 (data not shown). After electroblotting and immunodetection, it was possible to distinguish the different PBPs. In male extracts, the mixture of the anti-Mbra-1 and anti-Mbra-2 antibodies stained strongly the two spots with pI=5.0 and pi=5.3
(Fig. 2c). The anti-Mbra-1 and anti-Mbra-2 antibodies alone stained the spots at pi=5.3 and pI=5.0 respectively (Fig. 2c). The same staining was observed in the female extracts. The isoelectric points of the PBPs of different species of Lepidoptera were calculated on the basis of amino acid composition, or measured (for A. polyphemus, Manduca sexta and Bombyx mori) within a narrow range, between 4.43 and 5.12 (Pelosi and Maida, 1995): our estimated pI are in agreement with these data. Two-dimensional electrophoresis provided a better separation of the proteins Mbra-1 and Mbra-2 in male and female homogenates than after native-PAGE, and showed some qualitative differences between the two sexes: fewer proteins were visualized in the female homogenates, and the spot containing the Mbra-! is less abundant in the female extracts. However, the proteins with the SKELI sequence (Mbra-1 and M b r a - l ' ) appear to have the same isoelectric point and their separation may need a higher resolution system, such as a Rotofor (BioRad; Prestwich, 1993), which generates a wider pH gradient.
Pheromone binding study After incubation of the 3H-Zll-16:Ac with the antennal homogenates from males and females or with the purified male PBPs, the protein-bound pheromone was analyzed by native-PAGE and fluorography (Fig. 3). In male antennal homogenates, the binding was localized only in the region of migration of the PBPs and no radio-
9 A~¢~
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(b)
PBPs
FIGURE 1. Non-denaturing polyacrylamidegel electrophoresis (native-PAGE, 16.8% acrylamide) of crude extracts from different tissues of male and female M. brassicae: head (1), thorax (1), antennae (50), legs (6), abdomen (3 segments) and 10 ~1 of female hemolymph. (a) Proteins were visualized by Coomassie blue staining (solution of 0.035% Blue-R in 12% trichloroacetic acid, 5% ethanol). (b) Western-blots of the same samples (10% of each sample) with primary anti-Mbra-1 and antiMbra-2 antibodies mixed (dilution 1:500, detection by chemiluminescence). Arrows indicate the positions of the PBPs and the GOBP2 in antennal extracts.
PHEROMONE-BINDING PROTEINS OF MAMESTRA BRASSICAE
(b) Detail of Coonmssie Staining
(a) Coomassie Blue Staining
pH
6,0
5,3 5,0
217
5,3 5,0
4,0
(c) Details of ECL Mixture of anti-Mbra-1 and anti-Mbra-2 antibodies
Anti-Mbra- 1 antibodies Anti-Mbra-2 antibodies
,1
5,35,0
5,3
5,0
FIGURE 2. Two-dimensional electrophoresis of antennal homogenates of male M. brassicae. The pH range of the first dimension gel is indicated under the gels and the position of some molecular weight standards (kDa) used in the second dimension gel (tricine SDS-PAGE) are indicated at the left of the gels. (a) Coomassie blue staining of male antennal extracts. (b) Detail of Coomassie staining, with the two principal spots in which several proteins were resolved. (c) Western-blots of male antennal extracts with details of ECL (use of the anti-Mbra-1 and anti-Mbra-2 antibodies mixed or separately).
(b) Autoradiography
(a) Coomassie Blue Staining
d
o,
S
FIGURE 3. Binding on gel between 3H-Z11-16:Ac and OBPs. Homogenates of M. brassicae antennae or purified Mbra-PBPs were incubated with the tritiated pheromone and electrophoresed. (a) Coomassie blue staining of crude antennal extracts and purified proteins, showing the positions of the PBPs and GOBP2 and their amounts. (b) Fluorography and autoradiography of the same samples showing the tritiated pheromone associated with the different proteins.
218
MAIBI~CHE-COISNE MARTINE et al.
activity was found associated with the third band corresponding to the GOBP2. In the female homogenates, the band 2 (Mbra-l: SKELI) and particularly the band 3 (GOBP2) were labelled. Only the male purified proteins Mbra- 1 and Mbra- 1 ', with the SKELI N-terminus, bound the pheromone in our experimental conditions and no binding was observed with the protein Mbra-2 (SQEIM). The sample preparation and the HPLC apparently did not alter the binding properties of the proteins. In addition, the binding between the proteins and the pheromonal compound appeared strong enough to remain stable during electrophoresis.
DISCUSSION The polyclonal antibodies raised against the PBPs of M. brassicae showed that these proteins are only present
in both male and female antennae and share this tissuespecificity with all other PBP studied in Lepidoptera (Vogt and Riddiford, 1981; Gy6rgyi et al., 1988; Vogt et al., 1989; Maida et al., 1993). No cross-reactivity was found with the GOBP2 (band 3) in the antennal extracts, which confirms the specificity of the antibodies tested. The faint staining observed only in the female legs is interesting and could be interpreted as a cross-reactivity with a protein of lower mobility which shares common epitopes with the PBPs. In Drosophila melanogaster, which has chemoreception sensory hairs in its legs like other Diptera, in situ hybridization between putative OBP clones and legs tissues has been reported (Pikielny et al., 1994), and recent data have indicated the existence of a putative odorant-binding protein in the antennae and legs of a Phasmid (Tuccini et al., 1996). Two-dimensional electrophoresis coupled with Western-blotting revealed additional proteins than previously detected, with a molecular weight (14-16 kDa) and an isoelectric point (4.5-6.0) alone compatible with PBP and GOBP. The use of the two antibodies raised against the PBP allowed us to separate clearly the Mbra- I' from the Mbra-2 which co-elute together in native-PAGE. In the males, the SKELI sequence was found in two distinct bands after native-PAGE (Mbra- 1 and Mbra- 1'), which could indicate either two different proteins with the same N-terminus or two forms of the same amino acid sequence with different arrangements of disulfide bridges, as assumed for the PBP of A. polyphemus by Ziegelberger (1996). After two-dimensional electrophoresis and MALDI-TOFMS (matrix assisted laser desorption/ionization-time of flight mass spectrometry, Blais et al., 1996), it is impossible to conclude because Mbra-I and Mbra-l' have close isoelectric points and close molecular weights (16 200 and 16 180 Da respectively). Separations using a Rotofor (Bio Rad) system are in progress (Maida et al., personal communication) to clarify this point. The study of binding between the tritiated major pheromonal compound and the antennal homogenates or pur-
ified PBPs of M. brassicae revealed important differences in the functional role of these proteins, as follows. Female extracts
The binding intensity is higher with the band 3 (GOBP2) than with the bands 1 and 2 (PBPs). In A. polyphemus, a binding between a protein belonging to the GOBP family and the pheromone component has been shown in the antennal extracts of female (Vogt and Riddiford, 1981) and male (Ziegelberger, 1996). However, in our species, the binding with the GOBP2 did not occur in male homogenate where PBP and GOBP are probably in competition. In this case, the affinity of the male PBPs for the Z l l - 1 6 : A c seems higher than those of the GOBP2. In the female extracts the PBPs are in smaller amounts than the GOBP2 and the Mbra-1' is absent. So, there are two hypotheses concerning the binding by the GOBP2 in the female extract. Either it could be the result of the small amount of Mbra-I present in the female, which explains the repartition of Z I 1-16:Ac between the two proteins. Or it could indicate that the female Mbra1 and the male Mbra-1 have different affinities for the Z11-16:Ac, so they could probably be two different isoforms of the same protein. Nevertheless, such a competition in the same sensilla in vivo appears uncertain: indeed, it has been demonstrated in several moth species that the GOBPs are only localized in sensilla basiconica and not co-expressed with the PBPs in sensilla trichodea (Laue et al., 1994; Steinbrecht et al., 1995). The binding between a GOBP2 and a sex pheromone is in agreement with the assumption of broad binding properties of this class of proteins (Vogt et al., 1991 a). The major point of interest is the binding observed between the Mbra-1 of the female homogenate and the pheromone component. It is the first time that such a binding has been observed in a female, but it has to be confirmed with the purified protein. In general, female moths have been viewed as anosmic towards their own pheromone. However, the presence of PBP in a female has been shown in some Lepidoptera species, such as Bombyx mori (Vogt et al., 1991a; Maida et al., 1993) and Manduca sexta (Gy6rgyi et al., 1988), suggesting that females may smell pheromone. Recently female perception of the conspecific pheromone was demonstrated in Spodoptera littoralis (Ljtinberg et al., 1993). Male extracts
The Mbra-1 and Mbra-1 ', which bind the major compound of the sex pheromone, are the most abundant PBPs present in the male antennal extract as established after HPLC and quantification (Bio-Rad protein Assay, data not shown). The hypothesis that each insect pheromone component could have a unique high-affinity PBP has been previously suggested (Du and Prestwich, 19951, supported by the presence of multiple PBPs in several moth species: two different PBPs have been previously identified in Antheraea pernyi and Lymantria dispar (Raming et al., 1990; Krieger et al., 1991; Vogt et al.,
PHEROMONE-BINDINGPROTEINS OF MAMESTRABRASSICAE 1989). In L. dispar, the binding between the two PBPs present in the sensillar extract with the two enantiomers of disparlure showed that one of the two proteins (Ldis2) has a stronger affinity than the other for both enantiomers, and it has been suggested that the other one (Ldis1) could specifically bind another component of the pheromone (Vogt et al., 1989) still unknown. On the other hand, the ligand specificity of the two recombinant PBPs from A. pernyi (Aper-1, Aper-2) and the recombinant PBP of A. polyphemus (Apol-3) for two pheromonal components were previously determined (Du and Prestwich, 1995): the two recombinant PBPs of the same species showed opposite binding specificity for two different ligands, whereas Aper-1 and Apol-3 shared high sequence homology and exhibit both a high affinity to the same pheromone compound. These authors suggest that the ligand specificity is probably encoded in the primary structure of the proteins, which have evolved to bind unique ligands. Our results show that two native purified PBPs of the same species exhibit opposite affinities for its major pheromone compound, and are consistent with the data obtained with recombinant proteins. These results of binding lead to several hypotheses to account for the functional role of the PBPs in Mamestra brassicae, according to the data known about the composition of the pheromone and the sensory encoding in this species, in relationship with the different morphological types of sensilla characterized (Fig. 4). We can propose that in M. brassicae male antennae, the Mbra-1 and Mbra-1' occur in the long sensory hairs, where they bind the Zll-16:Ac, which is known to stimulate one of the two receptor cells (ORN A). The Mbra-2 could be devoted to the binding of other pheromone components (intra- or interspecific ones). This protein could be either coexpressed in the long sensilla trichodea (where the second ORN B is equally tuned to Z9-14:Ac and to Z1 lL o n g sensilla trichodea
Z9-14:Ae Zll-16:OH
1-16:Ac~
ra-1
i
'
Mbra-1 ? Mbra-2 ?
219
16:OH, two compounds that decrease the flight response of males to pheromone sources), or only expressed in the short sensilla trichodea (which ORN respond to the Z914:Ac, antagonist and the Zll-16:AId, synergist at 0.1%). Immunohistological studies with the monoclonal antibodies raised against the different PBPs of M. brassicae are in progress to clarify these hypothesis. Moreover, the protein Mbra-2 shares a high N-terminus homology (88%) with the PBP of another noctuid moth, Heliothis virescens, whose major pheromonal compound is the Z11-16:Aid (Krieger et al., 1993): this component also is a pheromonal compound in M. brassicae and could be a putative ligand for Mbra-2. In addition, the PBP of two sibling species, Spodoptera descoinsi and S. latifascia (Nagnan-Le Meillour et al., in prep.) crossreacted with the anti-Mbra-2, and their N-terminus was determined to belong to the type Mbra-2 (SQELMVKM). These two species use the Z9-14:Ac as a component of the pheromone, and it could be a ligand for the Mbra-2 too. It would be of great interest to determine what kind of PBP is present in the species using Z11-16:Ac in their pheromone. Nevertheless, in closely related species, a structure-function relationship for the PBPs can be established. In Noctuidae, the detection of heterospecific pheromone compounds was clearly established (Lucas and Renou, 1989). The presence of ORN tuned to heterospecific pheromone compounds could be correlated at the level of perireceptor events to the expression of specific PBPs, which might be selected to recognize molecules that do not belong to the species. This adaptive mechanism could participate in the premating isolation between sympatric species, which are in competition for resources.
Short sensilla trichodea
ORN C Z9-14:Ae e Z11-16:Ald
ORN D .9
Mbra-1 ? Mbra-2 ?
I j l i Q I o° H'llUUlol,glml..imlnlUNiNglll''ll
FIGURE4. Hypotheticalmodel for the pheromonalencoding in Mamestra brassicae. ORN, olfactoryreceptor neuron; +, activator; -, inhibitor; Zll-16:Ac=ll-cis-hexadecenyl acetate; Zll-16:Ald=ll-cis-hexadecenyl aldehyde; Z9-14:Ac=9-cistetradecenyl acetate; ZI 1-16:OH=I1-cis-hexadecenol. It is possible to associate ORN A with the PBPs Mbra-I and Mbra-l' and with the majorcomponentof the pheromone,the Z11-16:Ac. Other associationsare still hypothetical.
220
MAiBI~CHE-COISNE MARTINE et al.
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Acknowledgements--The authors are grateful to Mafie-Th6r~se Bethenod for technical assistance. P.N.L.M. is indebted to Glenn Prestwich for constant encouragement. This work is dedicated to Charles Descoins. Research supported by INRA. We thank Dick Vogt, Michael Burnet, Paolo Pelosi and an anonymous reviewer for helpful comments on the manuscript.