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Olfactory soluble proteins of cockroaches
Insect Biochemistry and Molecular Biology 29 (1999) 973–978 www.elsevier.com/locate/ibmb
Olfactory soluble proteins of cockroaches Jean-Franc¸ois Picimbon 1, Walter Soares Leal * Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science, 1–2 Ohwashi, Ibaraki, Tsukuba 305-8634, Japan Received 28 August 1998; received in revised form 15 June 1999; accepted 23 June 1999
Abstract We have characterized antennae-specific proteins from three species of cockroaches Periplaneta americana, P. fuliginosa and Blattella germanica. Based on the N-terminal sequences, cockroach antennal proteins can be divided into three groups: (i) proteins with amino acid similarity to OS-D, a product of gene cloning from Drosophila melanogaster, (ii) proteins similar to a phasmid, Sipyloidea sipylus, olfactory protein, and (iii) cockroach specific proteins without any relevant similarity to other known proteins. Whereas most of the antennae-specific proteins were detected in extracts from male and female antennae, some proteins are sex specific. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Periplaneta americana; Periplaneta fuliginosa; Blattella germanica
1. Introduction Chemical communication in cockroaches is achieved with the use of pheromones of remarkable structural diversity. These semiochemicals are divided into two broad classes: cuticular and volatile compounds, the former elicit sexual responses only after both sexes make physical contact, whereas the latter act over a distance. In some species males produce a long range sex pheromone that attracts females, but, as a rule, it is the females that do the luring (Charlton et al., 1993). Volatile female-released sex pheromones have been identified in Periplaneta americana as a mixture of periplanones B and A (Persoons et al., 1982; Hauptmann et al., 1986; Kitahara et al., 1987; Kuwahara and Mori 1989, 1991), whereas P. fuliginosa has been reported to utilize periplanone D (Takahashi et al., 1995). On the other hand, the sex pheromone for the brownbanded cockroach, Supella longipalpa has been identified as 5-(2⬘R,4⬘Rdimethylheptanyl)-3-methyl-2H-pyran-2-one (Charlton et al., 1993; Leal et al., 1995), which has no structural similarities to the germacrene-related periplanones. In * Corresponding author. Tel.: +81-298-38-6213; fax: +81-298-386028. E-mail address: [email protected] (W.S. Leal) 1 Present address: Institute of Physiology, Hohenheim University, Gartenstrasse 30 Stuttgart 70593, Germany
addition, the German cockroach, Blattella germanica, does release a volatile sex pheromone (Liang and Schal, 1993), which seems to belong to yet another group of compounds. If odorant binding proteins (OBPs) are involved in discrimination of chemical signals their specificity to the cognate ligands may be reflected in their primary structures. This correlation between OBPs and odorant molecules may not be realized in moth pheromone binding proteins (PBPs) because lepidopteran pheromones have little structural diversity. However, scarab beetles, which utilize lactone pheromones, have PBPs with low amino acid identity to the lepidopteran PBPs (Wojtasek et al., 1998), suggesting a relationship between pheromone structures and amino acid sequences of PBPs (Leal et al., 1998). The diversity of cockroach pheromones provides an interesting model to address the question of the correlation between OBPs and odorant molecules and allow us to get a better insight in the role of molecular filters postulated for these proteins. As the first step of these studies, we have characterized olfactory soluble proteins of three species of cockroaches, P. americana, P. fuliginosa and B. germanica. As we report here, antennae-specific proteins of cockroaches are remarkably different from lepidopteran OBPs.
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J.-F. Picimbon, W.S. Leal / Insect Biochemistry and Molecular Biology 29 (1999) 973–978
2. Materials and methods
2.4. Amino acid sequence analysis
2.1. Preparation of the protein extracts
Twenty antennae equivalent extracts from P. fuliginosa were required to sequence the four proteins of interest. B. germanica proteins were blotted for sequencing from 400 antennae-equivalents samples while P. americana were blotted from eight antennae-equivalents. For the sequencing of immobilized proteins, protein bands were directly placed in the cartridge of a gas phase sequencer (Beckman LF 3000 PS and Hewlett Packard 241 Protein Sequencer) without any further treatments. Sequence determination was repeated twice on samples obtained from different blots. The N-terminal sequences obtained were compared to proteins in the SwissProt data base.
Antennae of laboratory-raised cockroaches were excised at the base, lyophilized and extracted immediately or stored at ⫺80°C. The crude protein extracts were prepared by homogenization in 2 mM Tris–HCl, pH 7.5, 0.5 mM EDTA (Tris–EDTA buffer) using a glass hand homogenizer and centrifugation at 12,000g for 5 min at 4°C. In a typical experiment, 5 antennae from P. americana, 10 antennae from P. fuliginosa, 100 antennae from B. germanica were homogenized in 750 µl of Tris–EDTA buffer. Similar protocols were followed for preparing extracts from other body parts, particularly legs used as control.
3. Results 2.2. Electrophoresis and protein analysis 3.1. Periplaneta americana Electrophoresis in non-denaturing conditions was run on 15% polyacrylamide gels using a Bio-Rad gel. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. Antennal proteins were selected according to their size, electrophoretic mobility and tissue or sex specificity. 2.3. Electroblotting After electrophoretic separation in non-denaturing conditions, proteins were electroblotted to a glass-fiber (GF) filter, according to Aebersold et al. (1986). In brief, GF filters were pre-treated with trifluoroacetic acid (TFA) and dried. Immediately after the electrophoresis, the gel was soaked in the blotting solution (1% acetic acid in water) for at least 20 min. Meanwhile, the ‘sandwich’ for electroblotting was assembled. The sandwich consisted of sponge, Whatman filter paper, TFA-activated GF disk, the gel, Whatman filter paper and sponge, all humidified with blotting solution and oriented from the negative to the positive pole of the blotting buffer chamber. The sandwich was placed into the blotting chamber, which was filled with blotting buffer equilibrated at 0–4°C. Blotting was performed at 350 mA corresponding to 40 V for 3 h in an ice-bath (0–4°C). Proteins that had been electroblotted onto GC filters were detected by staining with Coomassie Brilliant Blue. The GF disk was soaked in staining solution (0.5% Coomassie Blue, 30% isopropyl alcohol and 10% acetic acid in water) for 2 min and then destained by shaking the blot in an aqueous solution containing 16.5% methanol/5% acetic acid several times. After rinsing twice with nanopure water to remove all traces of acid, protein bands of interest were cut out from the GF disk and stored at ⫺20°C prior to sequencing.
Electrophoretic analysis of soluble proteins from male and female antennae and legs of P. americana showed eight bands which appeared specific or more strongly expressed in antennal tissues. These proteins were called Pam1, 2, 3, 4, 5, 6, 7 and 8 in the order of mobility, 1 being the fastest migrating (Fig. 1). Their acidic nature, indicated by their mobility, is similar to that of the OBPs described to date. Proteins were blotted from the native gels to determine their N-terminal amino acid sequences by direct Edman degradation. As for other insect OBPs identified to date, N-terminal ends were not blocked. Pam8 was specific to male antennae; its sequence was identical to
Fig. 1. Electrophoretic comparison on a native 15% gel of proteins obtained from extracts of leg and male and female antennae of P. americana. L: posterior legs (1.5 leg-equivalents); FA: female antennae, and MA: male antennae (5 antennae-equivalents).
J.-F. Picimbon, W.S. Leal / Insect Biochemistry and Molecular Biology 29 (1999) 973–978
Pam7, which was expressed in both sexes (Figs. 1 and 2). Pam7 from male (Pam7m) and females (Pam7f) differed mainly in the first residues at the N-terminus (Fig. 2). Pam6 was also expressed strongly in males and females. Based on the N-terminal sequence, Pam6 was found to be related to a faster migrating protein from P. americana antennae (see below). Pam6 of males (Pam6m) and females (Pam6f) showed identical N-terminal amino acid sequences (Fig. 2). On the other hand, Pam5 of males (Pam5m) and females (Pam5f) showed significant difference in their N-terminus (Fig. 2). Pam4 and Pam3 were expressed in low amounts, Pam4 being expressed in both sexes while Pam3 was male-specific. Pam2 had different sequences in males and females while the fastest migrating protein, Pam1 was found equally in both sexes. 3.2. Periplaneta fuliginosa
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Fig. 3. Electrophoretic comparison on a native 15% gel of antennal protein extracts from P. fuliginosa. FA: female antennae; MA: male antennae (10 antennae-equivalents); L: posterior legs (2.5 legequivalents).
Samples from B. germanica required a larger amount of tissue to be visualized in the gel; the native polyacrylamide gel profile (100 antenna-equivalents and 80 leg-
equivalents) suggested the occurrence of four antennaespecific proteins, BG1, BG2, BG3, and BG4 (Fig. 4). Since we are interested in OBPs we did not investigate in further detail proteins clearly detected in control tissues (legs). Analysis of more concentrated extracts (200 legequivalents) revealed the occurrence of trace amounts of these four proteins in legs. Under our electrophoretic conditions, BG4 comigrated with Fuligi4 and Pam7, but the sequence of BG4 from female (BG4f) showed lower amino acid similarity to Fuligi4 (39%) than to Pam7 (66%) (Fig. 5). While it was not possible to get a reliable sequence for BG3, sequence from the band containing BG1 and BG2 gave the following sequence: HTEE-IKELEKK-QDKHV. Given the fact that this sequence was not obtained from a single band and that trace amounts were also detected in legs, BG1 and BG2 were not considered as a putative OBPs in the following discussion. Further analysis of more concentrated extracts (400 antenna-equivalents) showed the occurrence of three acidic proteins (BG⬘1, BG⬘2 and BG⬘3) that were found
Fig. 2. Comparison between sexes of N-terminal amino acid sequences of proteins expressed in P. americana antennae. Protein sequences are ordered according to the pattern of migration on a 15% native gel. Pam8 and Pam3 were detected only in male antennae.
Fig. 4. Electrophoretic comparison on a native 15% gel of antennal protein extracts from B. germanica. FA: female antennae; MA: male antennae (100 antennae-equivalents); L: posterior legs (80 legequivalents).
In P. fuliginosa, the native PAGE pattern gave four major bands (Fuligi1, 2, 3 and 4, according to migration) from the antennal extracts with no apparent differences between male and female samples (Fig. 3). Fuligi4 was highly expressed in antennal tissues, but was also found in legs. This protein is similar to two other proteins detected in P. americana, Pam8 and Pam7. Fuligi3 was the most abundant protein in antennae but was also detected in low amounts in leg extracts. This protein showed considerable similarity to Pam6 (78% identity) and Pam2 (50%) from P. americana. The fast migrating and antennae-specific proteins, Fuligi2 and Fuligi1, were not sex-specific. 3.3. Blattella germanica
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Fig. 5. Percentage identities based on N-terminal sequences of cockroach OBP-like proteins and their counterparts. Abbreviations: Fuligi, P. fuliginosa; Pam, P. americana; BG, B. germanica; OS-D, Drosophila protein; Ecalc, Eurycantha calcarata; Etiar, Extatosoma tiaratum; Cmoro, Carausius morosus; Ccact, Cactoblastis cactorum; Ssipy, Sipyloidea sypilus.
in the dye front (data not shown). BG⬘m2 was malespecific and similar to Pam1 and Fuligi1, whereas BG⬘m1 (VETPALKEDKD) showed no similarity to other known proteins. On the other hand, BG⬘3 was found in both sexes, but the scanty amount of material was enough only to obtain a short N-terminal sequence from male extract.
4. Discussion We have identified soluble proteins expressed in the antennae of three species of cockroaches, P. americana, P. fuliginosa and B. germanica. In P. americana, Pam8, Pam7f, and Pam7m showed significant amino acid identity to the Drosophila melanogaster OS-D protein (McKenna et al., 1994; A-10 according to Pikielny et al., 1994) (Figs. 5 and 6). Also, Pam7m and Pam7f showed significant amino acid similarity to a protein (p10) that increases in nymphal regenerating legs of P. americana (Nomura et al., 1992). Recently, immunoblotting analysis revealed that p10 is expressed not only in the regenerating legs, but also in the antennae and heads of nymphs and adult cockroaches (Kitabayashi et al., 1998). On the other hand, Pam6 was found to be related to a protein previously described in the phasmid Sipyloidea sypilus, which has no appreciable sequence similarity to any known OBP or to other phasmid soluble proteins (Mameli et al., 1996). Pam5f showed amino acid identity to OS-D (35%), but Pam5m was not related to any known proteins. Pam4 seems to be antennae-specific and the same type is present in both sexes but low amounts of Pam4 prevented the identification of a longer
Fig. 6. Classification of cockroach olfactory soluble proteins according to their identity to Drosophila OS-D (A), S. sypilus OBP-like protein (B), and cockroach-specific proteins having the sequence DXLDDDTK (⫺) or EPGXVG-XT (D). Abbreviations: Fuligi, P. fuliginosa; Pam, P. americana; BG, B. germanica; OS-D, Drosophila protein; Ecalc, Eurycantha calcarata; Etiar, Extatosoma tiaratum; Cmoro, Carausius morosus; Ccact, Cactoblastis cactorum; Ssipy, Sipyloidea sypilus.
sequence. Pam3 is similar to other cockroach proteins, but not to any known OBPs. Pam2 is similar to a putative olfactory protein from S. sypilus, while Pam1 belongs to the class of cockroach specific proteins (Fig. 6). Fuligi4 from P. fuliginosa showed considerable amino acid identity to the Drosophila OS-D protein (McKenna et al., 1994) and moderate similarity to the sub-class of 14–15 kDa OS-related phasmid proteins, particularly Extatosoma calcarata, Extatosoma tiaratum, and Carausius morosus OBPs (Mameli et al., 1996). On the other hand, Fuligi3 showed 50% identity to an olfactory protein from S. sypilus. Fuligi2 and Fuligi1 are cockroachspecific proteins related to other proteins found in P. americana and B. germanica. B. germanica BG4f showed significant amino acid identity to the Drosophila OS-D and phasmid proteins, especially the one isolated from C. morosus. Based on their N-terminal amino acid sequence and their amino acid identity to phasmid and Drosophila proteins, antennal proteins from cockroaches can be divided into four classes (Fig. 6). The first class contains proteins similar to OS-D protein. OS-D related proteins have been previously detected in three species of phasmids, Eurycantha calcarata, Extatosoma tiaratum and Caraus-
J.-F. Picimbon, W.S. Leal / Insect Biochemistry and Molecular Biology 29 (1999) 973–978
ius morosus. We showed here the presence of such proteins in three species of cockroach, Pam8m/Pam7m/Pam5f (P. americana), Fuligi4 (P. fuliginosa) and BG4f (B. germanica) (Fig. 6). A member of this class was also found in Blatta orientalis (Mameli, 1997) and two species of bees (Picimbon and Leal, unpublished data), suggesting that OS-D like proteins occur not only in cockroaches but also in phyllogenetically distant insects. Similar proteins were also found in the sensory organs (legs and labrum) of Schistocera gregaria (AF070961-5; Pelosi and Angeli, personnal communication). Another similar protein was reported in the palpi of a lepidopteran species, Cactoblastis cactorum (Maleszka and Stange, 1997). Since the palpi are involved in CO2 detection, and given the fact that OSD is found in phyllogenetically distinct orders, OS-D has also been suggested to play a role in CO2 detection (Maleszka and Stange, 1997). The second class included proteins with high amino acid identity to the protein purified from S. Sipylus antennae: Pam2f, Pam6m, Fuligi3. These proteins were found to have no similarity to other phasmid proteins or other known insect OBPs (Mameli et al., 1996). Furthermore, S. sipylus is phyllogenetically distant to E. calcarata, E. tiaratum and C. morosus, which share another type of OBP-like protein. No similarity was found with phasmid proteins grouped as 18–19 kDa and OBP-like proteins (Mameli et al., 1996). S. sipylus OBP-like related proteins are very concentrated in the antennal extracts and their levels are similar in males and females. N-terminal sequences obtained for Pam6f and Pam6m are identical, showing that the same protein is expressed in both sexes. Furthermore, Pam2 which showed 57% amino acid identity to Pam6 would constitute a second type of S. sipylus OBP-like protein, characterized by its small molecular weight and higher acidity. Proteins of the third class share about 50% identity between each other, in particular a DXLDDDTK sequence. These antennae-specific proteins are fast migrating proteins on native polyacrylamide gels; they migrate in the zone corresponding to small and acidic OBPs. The other minor proteins specific to the antennae can be grouped in a fourth class based on conserved EPGXVG-XT sequence. They migrate in the same zone as the proteins of the third class and according to their size, mobility and tissue-specificity, they may also be considered as putative olfactory proteins. These proteins were also found in Periplaneta spp. and B. germanica. Interestingly, the above mentioned proteins are not sex-specific and are found at the same level in males and females. Even if these proteins are involved in the early olfactory processing as odorant-binding proteins, the lack of sex specificity is not entirely surprising since besides sex pheromones, cockroaches rely on a wide range of volatile chemicals for location of food sources.
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In addition, we also observed the presence of male-specific antennal proteins, such as Pam8m and Pam3m, which were highly expressed in the male antennae of P. americana, but could not be detected in female extracts of the same or higher concentrations. In addition, expression of Pam8 seems to be under a physiological control in adults since it has not been observed in samples collected from old males. In P. fuliginosa, one protein comigrating with Fuligi4 was also detected in male antennal extracts. Although this is only indirect evidence, the expression of these proteins only in male antennae suggests a role in the recognition of femalereleased sex pheromones. Whether the antennal proteins described here function as odorant-binding proteins, as we suggest, requires the additional demonstration that they are expressed specifically in olfactory sensilla and that they indeed bind to odorant or pheromone molecules.
Acknowledgements Supported in part by a program for promoting Basic Research Activities for Innovative Bioscience (BRAIN) and by a coordination fund for promoting science and technology by the Science and Technology Agency (STA) of Japan. We are grateful to Dr H. Saito (NISES) for his technical assistance in protein sequencing and the members of our laboratory for revising an earlier version of the manuscript.
References Aebersold, R.H., Teplow, D.B., Hood, L.E., Kent, S.B.H., 1986. Electroblotting onto activated glass: high efficiency preparation of proteins from analytical sodium dodecyl sulfate–polyacrylamide gels for direct sequence analysis. J. Biol. Chem. 261, 4229–4238. Charlton, R.E., Webster, F.X., Zhang, A., Schal, C., Liang, D., Sreng, I., Roelofs, W.L., 1993. Sex pheromone of the brownbanded cockroach is an unusual dialkyl-substituted α-pyrone. Proc. Natl. Acad. Sci. USA 90, 10202–10205. Hauptmann, H., Mu¨hlbauer, G., Sass, H., 1986. Identifizierung und synthese von Periplanon A. Tetrahedron Lett. 27, 6189–6192. Kitabayashi, A.N., Arai, T., Kubo, T., Natori, S., 1998. Molecular cloning of cDNA for p10, a novel protein that increases in the regenerating legs of Periplaneta americana (American cockroach). Insect Biochem. Molec. Biol. 28, 785–790. Kitahara, T., Mori, M., Mori, K., 1987. Total synthesis of (⫺)-periplanone B, natural major sex-excitant pheromone of the American cockroach Periplaneta americana. Tetrahedron 43, 1689–1699. Kuwahara, S., Mori, K., 1989. Clarification of the structure of Pearsoon’s periplanone A, an artifact derived from Hauptamnn’s periplanone A. Tetrahedron Lett. 30, 7447–7450. Kuwahara, S., Mori, K., 1991. Synthesis of both enantiomers of Hauptmann’s periplanone A and clarification of the structure of Persoons’s periplanone A. Tetrahedron 46, 8083–8092. Leal, W.S., Shi, X., Liang, D., Schal, C., Meinwald, J., 1995. Application of chiral gas chromatography with electroantennographic
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detection to the determination of the stereochemistry of a cockroach sex pheromone. Proc. Natl. Acad. Sci. USA 92, 1033–1037. Leal, W.S., Wojtasek, H., Picimbon, J.-F., Kuwahara, S., Saito, H., Hasegawa, M., 1998. Perireceptor events in pheromone perception in scarab beetles. J. Asia-Pacific Entomol. 1, 1–8. Liang, D., Schal, C., 1993. Volatile sex pheromone in the female German cockroach. Experientia 49, 324–328. Maleszka, R., Stange, G., 1997. Molecular cloning, by a novel approach, of a cDNA encoding a putative olfactory protein in the labial palps of the moth Cactoblastis cactorum. Gene 202, 39–43. Mameli, M., Tuccini, A., Mazza, M., Petacchi, R., Pelosi, P., 1996. Soluble proteins in chemosensory organs of phasmids. Insect Biochem. Molec. Biol. 26, 875–882. Mameli, M., 1997. Studio della chemiorecezione negli insetti: OBP e proteine solubili. PhD thesis. University of Pisa, Italy. McKenna, M.P., Hekmat-Scafe, D.S., Gaines, P., Carlson, J.R., 1994. Putative Drosophila pheromone-binding-proteins expressed in a subregion of the olfactory system. J. Biol. Chem. 269, 16340– 16347.
Nomura, A., Kawasaki, K., Kubo, T., Natori, S., 1992. Purification and localization of p10, a novel protein that increases in nymphal regenarting legs of Periplaneta americana (American cockroach). Int. J. Dev. Biol. 36, 391–398. Persoons, C.J., Verwiel, P.E.J., Ritter, F.J., Talman, E., Nooijen, P.J.F., Nooijen, W.J., 1982. Sex pheromone of the American cockroach, with emphasis on structure elucidation of periplanone-A. J. Chem. Ecol. 8, 439–451. Pikielny, C.W., Hasan, G., Rouyer, F., 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. Takahashi, S., Watanabe, K., Saito, S., Nomura, Y., 1995. Isolation and biological activity of the smoky brown cockroach, Periplaneta fuliginosa Serville (Dictyoptera: Blattidae). Appl. Entomol. Zool. 30, 357–360. Wojtasek, H., Hansson, B.S., Leal, W.S., 1998. Attracted or repelled?—A matter of two neurons, one pheromone binding protein and a chiral center. Biochem. Biophys. Res. Commun. 250, 217–222.
Olfactory soluble proteins of cockroaches Jean-Franc¸ois Picimbon 1, Walter Soares Leal * Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science, 1–2 Ohwashi, Ibaraki, Tsukuba 305-8634, Japan Received 28 August 1998; received in revised form 15 June 1999; accepted 23 June 1999
Abstract We have characterized antennae-specific proteins from three species of cockroaches Periplaneta americana, P. fuliginosa and Blattella germanica. Based on the N-terminal sequences, cockroach antennal proteins can be divided into three groups: (i) proteins with amino acid similarity to OS-D, a product of gene cloning from Drosophila melanogaster, (ii) proteins similar to a phasmid, Sipyloidea sipylus, olfactory protein, and (iii) cockroach specific proteins without any relevant similarity to other known proteins. Whereas most of the antennae-specific proteins were detected in extracts from male and female antennae, some proteins are sex specific. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Periplaneta americana; Periplaneta fuliginosa; Blattella germanica
1. Introduction Chemical communication in cockroaches is achieved with the use of pheromones of remarkable structural diversity. These semiochemicals are divided into two broad classes: cuticular and volatile compounds, the former elicit sexual responses only after both sexes make physical contact, whereas the latter act over a distance. In some species males produce a long range sex pheromone that attracts females, but, as a rule, it is the females that do the luring (Charlton et al., 1993). Volatile female-released sex pheromones have been identified in Periplaneta americana as a mixture of periplanones B and A (Persoons et al., 1982; Hauptmann et al., 1986; Kitahara et al., 1987; Kuwahara and Mori 1989, 1991), whereas P. fuliginosa has been reported to utilize periplanone D (Takahashi et al., 1995). On the other hand, the sex pheromone for the brownbanded cockroach, Supella longipalpa has been identified as 5-(2⬘R,4⬘Rdimethylheptanyl)-3-methyl-2H-pyran-2-one (Charlton et al., 1993; Leal et al., 1995), which has no structural similarities to the germacrene-related periplanones. In * Corresponding author. Tel.: +81-298-38-6213; fax: +81-298-386028. E-mail address: [email protected] (W.S. Leal) 1 Present address: Institute of Physiology, Hohenheim University, Gartenstrasse 30 Stuttgart 70593, Germany
addition, the German cockroach, Blattella germanica, does release a volatile sex pheromone (Liang and Schal, 1993), which seems to belong to yet another group of compounds. If odorant binding proteins (OBPs) are involved in discrimination of chemical signals their specificity to the cognate ligands may be reflected in their primary structures. This correlation between OBPs and odorant molecules may not be realized in moth pheromone binding proteins (PBPs) because lepidopteran pheromones have little structural diversity. However, scarab beetles, which utilize lactone pheromones, have PBPs with low amino acid identity to the lepidopteran PBPs (Wojtasek et al., 1998), suggesting a relationship between pheromone structures and amino acid sequences of PBPs (Leal et al., 1998). The diversity of cockroach pheromones provides an interesting model to address the question of the correlation between OBPs and odorant molecules and allow us to get a better insight in the role of molecular filters postulated for these proteins. As the first step of these studies, we have characterized olfactory soluble proteins of three species of cockroaches, P. americana, P. fuliginosa and B. germanica. As we report here, antennae-specific proteins of cockroaches are remarkably different from lepidopteran OBPs.
0965-1748/99/$ - see front matter. 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 0 7 3 - 9
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J.-F. Picimbon, W.S. Leal / Insect Biochemistry and Molecular Biology 29 (1999) 973–978
2. Materials and methods
2.4. Amino acid sequence analysis
2.1. Preparation of the protein extracts
Twenty antennae equivalent extracts from P. fuliginosa were required to sequence the four proteins of interest. B. germanica proteins were blotted for sequencing from 400 antennae-equivalents samples while P. americana were blotted from eight antennae-equivalents. For the sequencing of immobilized proteins, protein bands were directly placed in the cartridge of a gas phase sequencer (Beckman LF 3000 PS and Hewlett Packard 241 Protein Sequencer) without any further treatments. Sequence determination was repeated twice on samples obtained from different blots. The N-terminal sequences obtained were compared to proteins in the SwissProt data base.
Antennae of laboratory-raised cockroaches were excised at the base, lyophilized and extracted immediately or stored at ⫺80°C. The crude protein extracts were prepared by homogenization in 2 mM Tris–HCl, pH 7.5, 0.5 mM EDTA (Tris–EDTA buffer) using a glass hand homogenizer and centrifugation at 12,000g for 5 min at 4°C. In a typical experiment, 5 antennae from P. americana, 10 antennae from P. fuliginosa, 100 antennae from B. germanica were homogenized in 750 µl of Tris–EDTA buffer. Similar protocols were followed for preparing extracts from other body parts, particularly legs used as control.
3. Results 2.2. Electrophoresis and protein analysis 3.1. Periplaneta americana Electrophoresis in non-denaturing conditions was run on 15% polyacrylamide gels using a Bio-Rad gel. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. Antennal proteins were selected according to their size, electrophoretic mobility and tissue or sex specificity. 2.3. Electroblotting After electrophoretic separation in non-denaturing conditions, proteins were electroblotted to a glass-fiber (GF) filter, according to Aebersold et al. (1986). In brief, GF filters were pre-treated with trifluoroacetic acid (TFA) and dried. Immediately after the electrophoresis, the gel was soaked in the blotting solution (1% acetic acid in water) for at least 20 min. Meanwhile, the ‘sandwich’ for electroblotting was assembled. The sandwich consisted of sponge, Whatman filter paper, TFA-activated GF disk, the gel, Whatman filter paper and sponge, all humidified with blotting solution and oriented from the negative to the positive pole of the blotting buffer chamber. The sandwich was placed into the blotting chamber, which was filled with blotting buffer equilibrated at 0–4°C. Blotting was performed at 350 mA corresponding to 40 V for 3 h in an ice-bath (0–4°C). Proteins that had been electroblotted onto GC filters were detected by staining with Coomassie Brilliant Blue. The GF disk was soaked in staining solution (0.5% Coomassie Blue, 30% isopropyl alcohol and 10% acetic acid in water) for 2 min and then destained by shaking the blot in an aqueous solution containing 16.5% methanol/5% acetic acid several times. After rinsing twice with nanopure water to remove all traces of acid, protein bands of interest were cut out from the GF disk and stored at ⫺20°C prior to sequencing.
Electrophoretic analysis of soluble proteins from male and female antennae and legs of P. americana showed eight bands which appeared specific or more strongly expressed in antennal tissues. These proteins were called Pam1, 2, 3, 4, 5, 6, 7 and 8 in the order of mobility, 1 being the fastest migrating (Fig. 1). Their acidic nature, indicated by their mobility, is similar to that of the OBPs described to date. Proteins were blotted from the native gels to determine their N-terminal amino acid sequences by direct Edman degradation. As for other insect OBPs identified to date, N-terminal ends were not blocked. Pam8 was specific to male antennae; its sequence was identical to
Fig. 1. Electrophoretic comparison on a native 15% gel of proteins obtained from extracts of leg and male and female antennae of P. americana. L: posterior legs (1.5 leg-equivalents); FA: female antennae, and MA: male antennae (5 antennae-equivalents).
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Pam7, which was expressed in both sexes (Figs. 1 and 2). Pam7 from male (Pam7m) and females (Pam7f) differed mainly in the first residues at the N-terminus (Fig. 2). Pam6 was also expressed strongly in males and females. Based on the N-terminal sequence, Pam6 was found to be related to a faster migrating protein from P. americana antennae (see below). Pam6 of males (Pam6m) and females (Pam6f) showed identical N-terminal amino acid sequences (Fig. 2). On the other hand, Pam5 of males (Pam5m) and females (Pam5f) showed significant difference in their N-terminus (Fig. 2). Pam4 and Pam3 were expressed in low amounts, Pam4 being expressed in both sexes while Pam3 was male-specific. Pam2 had different sequences in males and females while the fastest migrating protein, Pam1 was found equally in both sexes. 3.2. Periplaneta fuliginosa
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Fig. 3. Electrophoretic comparison on a native 15% gel of antennal protein extracts from P. fuliginosa. FA: female antennae; MA: male antennae (10 antennae-equivalents); L: posterior legs (2.5 legequivalents).
Samples from B. germanica required a larger amount of tissue to be visualized in the gel; the native polyacrylamide gel profile (100 antenna-equivalents and 80 leg-
equivalents) suggested the occurrence of four antennaespecific proteins, BG1, BG2, BG3, and BG4 (Fig. 4). Since we are interested in OBPs we did not investigate in further detail proteins clearly detected in control tissues (legs). Analysis of more concentrated extracts (200 legequivalents) revealed the occurrence of trace amounts of these four proteins in legs. Under our electrophoretic conditions, BG4 comigrated with Fuligi4 and Pam7, but the sequence of BG4 from female (BG4f) showed lower amino acid similarity to Fuligi4 (39%) than to Pam7 (66%) (Fig. 5). While it was not possible to get a reliable sequence for BG3, sequence from the band containing BG1 and BG2 gave the following sequence: HTEE-IKELEKK-QDKHV. Given the fact that this sequence was not obtained from a single band and that trace amounts were also detected in legs, BG1 and BG2 were not considered as a putative OBPs in the following discussion. Further analysis of more concentrated extracts (400 antenna-equivalents) showed the occurrence of three acidic proteins (BG⬘1, BG⬘2 and BG⬘3) that were found
Fig. 2. Comparison between sexes of N-terminal amino acid sequences of proteins expressed in P. americana antennae. Protein sequences are ordered according to the pattern of migration on a 15% native gel. Pam8 and Pam3 were detected only in male antennae.
Fig. 4. Electrophoretic comparison on a native 15% gel of antennal protein extracts from B. germanica. FA: female antennae; MA: male antennae (100 antennae-equivalents); L: posterior legs (80 legequivalents).
In P. fuliginosa, the native PAGE pattern gave four major bands (Fuligi1, 2, 3 and 4, according to migration) from the antennal extracts with no apparent differences between male and female samples (Fig. 3). Fuligi4 was highly expressed in antennal tissues, but was also found in legs. This protein is similar to two other proteins detected in P. americana, Pam8 and Pam7. Fuligi3 was the most abundant protein in antennae but was also detected in low amounts in leg extracts. This protein showed considerable similarity to Pam6 (78% identity) and Pam2 (50%) from P. americana. The fast migrating and antennae-specific proteins, Fuligi2 and Fuligi1, were not sex-specific. 3.3. Blattella germanica
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Fig. 5. Percentage identities based on N-terminal sequences of cockroach OBP-like proteins and their counterparts. Abbreviations: Fuligi, P. fuliginosa; Pam, P. americana; BG, B. germanica; OS-D, Drosophila protein; Ecalc, Eurycantha calcarata; Etiar, Extatosoma tiaratum; Cmoro, Carausius morosus; Ccact, Cactoblastis cactorum; Ssipy, Sipyloidea sypilus.
in the dye front (data not shown). BG⬘m2 was malespecific and similar to Pam1 and Fuligi1, whereas BG⬘m1 (VETPALKEDKD) showed no similarity to other known proteins. On the other hand, BG⬘3 was found in both sexes, but the scanty amount of material was enough only to obtain a short N-terminal sequence from male extract.
4. Discussion We have identified soluble proteins expressed in the antennae of three species of cockroaches, P. americana, P. fuliginosa and B. germanica. In P. americana, Pam8, Pam7f, and Pam7m showed significant amino acid identity to the Drosophila melanogaster OS-D protein (McKenna et al., 1994; A-10 according to Pikielny et al., 1994) (Figs. 5 and 6). Also, Pam7m and Pam7f showed significant amino acid similarity to a protein (p10) that increases in nymphal regenerating legs of P. americana (Nomura et al., 1992). Recently, immunoblotting analysis revealed that p10 is expressed not only in the regenerating legs, but also in the antennae and heads of nymphs and adult cockroaches (Kitabayashi et al., 1998). On the other hand, Pam6 was found to be related to a protein previously described in the phasmid Sipyloidea sypilus, which has no appreciable sequence similarity to any known OBP or to other phasmid soluble proteins (Mameli et al., 1996). Pam5f showed amino acid identity to OS-D (35%), but Pam5m was not related to any known proteins. Pam4 seems to be antennae-specific and the same type is present in both sexes but low amounts of Pam4 prevented the identification of a longer
Fig. 6. Classification of cockroach olfactory soluble proteins according to their identity to Drosophila OS-D (A), S. sypilus OBP-like protein (B), and cockroach-specific proteins having the sequence DXLDDDTK (⫺) or EPGXVG-XT (D). Abbreviations: Fuligi, P. fuliginosa; Pam, P. americana; BG, B. germanica; OS-D, Drosophila protein; Ecalc, Eurycantha calcarata; Etiar, Extatosoma tiaratum; Cmoro, Carausius morosus; Ccact, Cactoblastis cactorum; Ssipy, Sipyloidea sypilus.
sequence. Pam3 is similar to other cockroach proteins, but not to any known OBPs. Pam2 is similar to a putative olfactory protein from S. sypilus, while Pam1 belongs to the class of cockroach specific proteins (Fig. 6). Fuligi4 from P. fuliginosa showed considerable amino acid identity to the Drosophila OS-D protein (McKenna et al., 1994) and moderate similarity to the sub-class of 14–15 kDa OS-related phasmid proteins, particularly Extatosoma calcarata, Extatosoma tiaratum, and Carausius morosus OBPs (Mameli et al., 1996). On the other hand, Fuligi3 showed 50% identity to an olfactory protein from S. sypilus. Fuligi2 and Fuligi1 are cockroachspecific proteins related to other proteins found in P. americana and B. germanica. B. germanica BG4f showed significant amino acid identity to the Drosophila OS-D and phasmid proteins, especially the one isolated from C. morosus. Based on their N-terminal amino acid sequence and their amino acid identity to phasmid and Drosophila proteins, antennal proteins from cockroaches can be divided into four classes (Fig. 6). The first class contains proteins similar to OS-D protein. OS-D related proteins have been previously detected in three species of phasmids, Eurycantha calcarata, Extatosoma tiaratum and Caraus-
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ius morosus. We showed here the presence of such proteins in three species of cockroach, Pam8m/Pam7m/Pam5f (P. americana), Fuligi4 (P. fuliginosa) and BG4f (B. germanica) (Fig. 6). A member of this class was also found in Blatta orientalis (Mameli, 1997) and two species of bees (Picimbon and Leal, unpublished data), suggesting that OS-D like proteins occur not only in cockroaches but also in phyllogenetically distant insects. Similar proteins were also found in the sensory organs (legs and labrum) of Schistocera gregaria (AF070961-5; Pelosi and Angeli, personnal communication). Another similar protein was reported in the palpi of a lepidopteran species, Cactoblastis cactorum (Maleszka and Stange, 1997). Since the palpi are involved in CO2 detection, and given the fact that OSD is found in phyllogenetically distinct orders, OS-D has also been suggested to play a role in CO2 detection (Maleszka and Stange, 1997). The second class included proteins with high amino acid identity to the protein purified from S. Sipylus antennae: Pam2f, Pam6m, Fuligi3. These proteins were found to have no similarity to other phasmid proteins or other known insect OBPs (Mameli et al., 1996). Furthermore, S. sipylus is phyllogenetically distant to E. calcarata, E. tiaratum and C. morosus, which share another type of OBP-like protein. No similarity was found with phasmid proteins grouped as 18–19 kDa and OBP-like proteins (Mameli et al., 1996). S. sipylus OBP-like related proteins are very concentrated in the antennal extracts and their levels are similar in males and females. N-terminal sequences obtained for Pam6f and Pam6m are identical, showing that the same protein is expressed in both sexes. Furthermore, Pam2 which showed 57% amino acid identity to Pam6 would constitute a second type of S. sipylus OBP-like protein, characterized by its small molecular weight and higher acidity. Proteins of the third class share about 50% identity between each other, in particular a DXLDDDTK sequence. These antennae-specific proteins are fast migrating proteins on native polyacrylamide gels; they migrate in the zone corresponding to small and acidic OBPs. The other minor proteins specific to the antennae can be grouped in a fourth class based on conserved EPGXVG-XT sequence. They migrate in the same zone as the proteins of the third class and according to their size, mobility and tissue-specificity, they may also be considered as putative olfactory proteins. These proteins were also found in Periplaneta spp. and B. germanica. Interestingly, the above mentioned proteins are not sex-specific and are found at the same level in males and females. Even if these proteins are involved in the early olfactory processing as odorant-binding proteins, the lack of sex specificity is not entirely surprising since besides sex pheromones, cockroaches rely on a wide range of volatile chemicals for location of food sources.
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In addition, we also observed the presence of male-specific antennal proteins, such as Pam8m and Pam3m, which were highly expressed in the male antennae of P. americana, but could not be detected in female extracts of the same or higher concentrations. In addition, expression of Pam8 seems to be under a physiological control in adults since it has not been observed in samples collected from old males. In P. fuliginosa, one protein comigrating with Fuligi4 was also detected in male antennal extracts. Although this is only indirect evidence, the expression of these proteins only in male antennae suggests a role in the recognition of femalereleased sex pheromones. Whether the antennal proteins described here function as odorant-binding proteins, as we suggest, requires the additional demonstration that they are expressed specifically in olfactory sensilla and that they indeed bind to odorant or pheromone molecules.
Acknowledgements Supported in part by a program for promoting Basic Research Activities for Innovative Bioscience (BRAIN) and by a coordination fund for promoting science and technology by the Science and Technology Agency (STA) of Japan. We are grateful to Dr H. Saito (NISES) for his technical assistance in protein sequencing and the members of our laboratory for revising an earlier version of the manuscript.
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