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cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis
Comparative Biochemistry and Physiology Part C 134 (2003) 513–520
cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis Neil M. Parkinsona,*, Christine M. Conyersa, Jeff N. Keenb, Alan D. MacNicolla, Ian Smitha,1, Robert J. Weavera a Plant Health Group, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK School of Biochemistry and Molecular Biology, Astbury Building University of Leeds, Leeds LS2 9JT, UK
b
Received 9 October 2002; received in revised form 29 January 2003; accepted 12 February 2003
Abstract Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro antihaemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large ()30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: cDNA; Pimpla hypochondriaca; Parasitoid; Hymenoptera; Wasp; Venom; Laccase; Trehalase; vpr
1. Introduction Insects possess a potent cellular immune system which is able to encapsulate foreign materials, including parasitoid eggs implanted into the haemocoel of hosts (Salt, 1957; Strand and Pech, 1995; Schmidt et al., 2001; Stanley and Ho, 2001). *Corresponding author. Tel.: 44-1904-462268; fax.: 44-1904-462111. E-mail address: [email protected] (N.M. Parkinson). 1 Present address: Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
At the time of oviposition, adult female parasitoids inject their hosts with various secretions, which are derived from ovarian tissue and venom. Factors produced in the ovarian tract which prevent the host’s immune system from destroying parasitoid eggs include polydnaviruses, virus-like particles and proteins (Vinson and Scott, 1974; Rotheram, 1973; Webb and Luckhart, 1994). Aside from our work, with the pupal endoparasitoid Pimpla hypochondriaca, relatively few studies have described the contribution of parasitoid venom to host immune suppression (Osman,
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N.M. Parkinson et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 513–520
1978; Kitano, 1982; Tanaka, 1987) and still less is known about the nature of venom components, although two cDNAs encoding proteins found in the venom gland of Chelonus sp. have been reported (Jones et al., 1992; Krishnan et al., 1994). We have established that venom from P. hypochondriaca has potent effects against haemocytes maintained in culture, and prevents encapsulation responses when injected into the haemocoel (Richards and Parkinson, 2000; Parkinson et al., 2002a). Previously, we have described paralytic and cytotoxic factors in venom fractionated by gel filtration, in addition to identifying L-DOPA oxidising activity (Parkinson and Weaver, 1999). A cDNA encoding an arthropod-specific phenoloxidase, an enzyme usually found in haemocytes, has been cloned from a cDNA library made from the venom-synthesising gland (Parkinson et al., 2001). Most recently, we have characterised cDNAs encoding a venom serine protease and a reprolysintype protease by random sequence analysis of clones in this library (Parkinson et al., 2002a,b). We have now extended the cDNA library sequence analysis and determined the primary structure of a further five proteins larger than 30 kDa, and additionally, confirmed these as venom constituents by N-terminal sequence analysis. 2. Materials and methods 2.1. Cloning and sequencing of cDNAs Lambda ZAP clones from a previously constructed venom gland library (Parkinson et al., 2001) were converted to p-Bluescript plasmids by in vivo excision according to the supplier’s protocol (Stratagene). Ampicillin-resistant colonies were selected and used to make broth cultures from which phagemid DNA was purified. The 59 end sequence of 500 cDNA clones was obtained using an automated DNA sequencer (ABI Prism 377). cDNAs predicted to encode proteins with signal sequences were identified using the SignalP program (Nielsen et al., 1997; http:yy www.cbs.dtu.dkyservicesysignalP-2.0). Clones encoding sequences, which matched N-terminal sequence from venom protein were fully sequenced for both DNA strands. Sequences were aligned using the CLUSTALW program (Thompson et al., 1994).
2.2. Separation of venom proteins and N-terminal sequencing Venom was size-fractionated using high-resolution gel filtration chromatography as described previously (Parkinson and Weaver, 1999). Pooled gel filtration-separated venom from fractions 23– 26, 30–32, 32–35 and 44–45 was concentrated 10-fold using an ultrafiltration device (UltrafreeMC Millipore, Bedford, MA). Venom proteins were separated by SDS-PAGE using 8, 10 or 12% acrylamide gels, and protein masses were estimated by reference to standards of 97, 66, 45, 21 and 14 kDa (BioRad). Separated proteins were blotted onto PVDF membrane at 30 V for 16 h in Towbin buffer using a tank blotter, as described previously (Parkinson et al., 2002a). N-terminal sequences (at least six residues) of blotted proteins were determined by Edman degradation using an Applied Biosystems Procise 494 HT sequencer. 3. Results 3.1. cDNAs encoding venom proteins greater than 30 kDa Listed in Table 1 are the GenBank accession numbers for five cDNAs encoding venom proteins, all of which possess signal sequences that are highlighted in the full sequences shown in Figs. 1 and 2 Fig. 3 Fig. 4. The predicted post-signal peptide sequences from the cDNAs are indicated, along with the matching sequence determined for venom proteins by Edman degradation. Following blotting and staining, vpr1, vpr2 and lac1 stained well and could easily be related to individual abundant venom proteins reported previously (Parkinson et al., 2002a); vpr2 and tre1 proteins stained less well after blotting (data not shown). Also indicated in Table 1 are the masses of the proteins calculated from the deduced amino acid sequences, the mass estimated from SDS-PAGE gels, and the fractions of gel filtration-separated venom in which the proteins were identified. 3.2. Venom proteins 3.2.1. vpr1 and vpr3 A BLAST search of the GenBank database revealed that the vpr1 sequence shares 63% amino acid identity with the Pimpla venom protein vpr3. Alignment of these two sequences (Fig. 1) with
CDNA
Accession No.
Protein N-terminus
Predicted N-terminus
Calculated Mass Da
SDS-PAGE Mass Da
Gel Filtration Fractions
Homology
lac1 tre1 vpr1 vpr2 vpr3
AJ427356 AJ459958 AJ459813 AJ459811 AJ459812
DNDGIT GSIGHV EGAGFVL DAVNEG DSDIYL
DNDGIT GSIGHV EGAGFVL DAVNEG DSDIYL
74020 63413 32929 53263 32723
76000 61000 32000 57000 32000
23–26 32–35 30–32 30–32 44–45
laccases trehalases vpr3 Drosophila gene vpr1
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Table 1
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Fig. 1. Encoded sequence and alignment of vpr1 and vpr3. Signal sequences are italicised. Residues at the beginning of the mature proteins, which are identical to N-terminal sequence, determined for venom proteins found in gel filtration-separated venom fractions 30–32 (vpr1) and 44–45 (vpr3), are underlined.
ClustalW indicated that the sequences are most dissimilar in the N-terminal one-third of proteins. No other proteins in the GenBank database had close sequence similar to vpr1 or vpr3. Despite having very similar masses, vpr1 and vpr3 eluted in disparate fractions (30–32 and 44–45, respectively) of gel filtration-separated venom. 3.2.2. vpr2 The vpr2 sequence was most similar to a protein identified from the genome sequence of Drosophila melanogaster (accession number AAF46793; 37% amino acid identity), with the greatest sequence similarity occurring in the carboxyl half of the proteins (Fig. 2). The longest contiguous sequence identity was 14 residues (IFKGDLNYRKLLGD; vpr2 residues 399–412). The GenBank database search identified proteins containing this sequence that were derived from genes present in the human (NM_024573), nematode (NM_075624), and yeast (NC_001145) genomes, indicating that genes related to vpr2 occur in a wide range of phyla. No function has been determined for any of these proteins. 3.2.3. lac1 When the predicted sequence of lac1 was used to search GenBank, similarity was detected to laccases from several wood-rotting fungi and to six proteins of unknown function identified from the Drosophila genome sequence. The lac1 sequence was aligned (Fig. 3) with the Coprinus cinereus laccase and with a Drosophila gene prod-
uct (AAF 57331), the latter having the highest degree of sequence identity (46%) to lac1. The crystal structure of the laccase from the ink cap fungus Coprinus cinereus has recently been resolved (Ducros et al., 1998) and copper-binding regions have been identified. Alignment with the fungal laccase indicated that lac1 shares 27% residue identity and contains all three sequences that bind copper in this enzyme. A Cysteine-rich region containing six cysteine residues occurs towards the N-terminus in both lac1 and the Drosophila gene products, but is absent from the Coprinus sequence. 3.2.4. tre1 A BLAST search indicated that tre1 was similar to trehalases from a wide range of organisms, and most similar to a trehalase from the bean shaped accessory gland of the mealworm Tenebrio molitor (Takiguchi et al., 1992). The tre1 and mealworm cDNAs aligned closely (Fig. 4) and shared 49% amino acid identity. Underlined in the Figure are two sequences conserved in trehalases, P-G-G-RF-x-E-x-Y-x-W-D-x-Y and Q-W-D-x-P-x-wGAx-WwPASx-P, identified in the Expasy Prosite entry PDOC00717 (Falquet et al., 2002). 4. Discussion We have demonstrated the utility of random sequence analysis of a cDNA library in providing a rapid analysis of the genes that encode many of the components of a complex venom. This cloning
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Fig. 2. Encoded sequence of vpr2. The sequence is aligned with that of a Drosophila gene product having the protein database identifier CG11474. The signal sequence of vpr2 is italicised. Residues at the beginning of the mature protein, which are identical to the Nterminus of a venom protein found in gel filtration-separated venom fractions 30–32, are underlined.
approach could be more widely applicable to the study of other complex secretory systems where cDNAs encoding proteins of interest are relatively abundant. It is difficult to estimate the diversity of venom components from SDS-PAGE gels, as individual proteins can have variable mobility, depending on the degree of post-translational modification. The current study has confirmed that the complex protein profile seen in SDS-PAGE gels of Pimpla venom is due to the expression of a diverse array of venom-encoding genes. vpr1 and vpr3 both have a very similar signal sequence and shares 63% overall amino acid identity. While the genes encoding these two proteins are related, sequence dissimilarities suggest that they have diverged at an early period in the evolutionary history of P. hypochondriaca. A search of the GenBank database did not reveal any sequences that were closely similar to either vpr1 or vpr3, and these proteins might have become highly specialised. The vpr2 protein is similar to a gene product identified in Drosophila whose function has not yet been determined, and genes, encoding a related protein have been identified in the nematode Cae-
norhabditis elegans and the yeast Saccharomyces cerevisiae. The Drosophila vpr2 homologue does not contain a signal sequence and the venom protein must have been derived from a similar Pimpla gene through an acquisition of sequence encoding a signal peptide. Identification of the venom protein lac1 suggests that the L-DOPA oxidising activity in venom, fractionated by gel filtration (Parkinson and Weaver, 1999) may be mediated both by the previously described arthropod haemocyte-type venom phenoloxidase and by a laccase-type enzyme. In support of this, lac1 is found in gel filtration fractions, previously shown to contain L-DOPA oxidising activity. In this study, the absence of anti-haemocytic activity in gel filtration separated venom containing lac1 reported previously (Parkinson et al., 2002a), may be due to the presence of a phenoloxidase inhibitor. The six-cysteine residues toward the N-terminus of lac1 and the Drosophila homologue may form a distinct structural motif. Pimpla lac1 may be the first insect laccase to be cloned, and further studies are needed to define its activity. In haemolymph of lepidopteran pupae (the developmental stage which is parasitised by Pim-
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Fig. 3. Encoded sequence of lac1. The sequence is aligned with a Drosophila gene and the laccase from the fungus C. cinereus, which have the protein database identifiers AAF 57331 and AAD 30964, respectively. The lac1 signal sequence is italicised and the start of the mature protein is indicated with an arrow. Shaded residues immediately following the signal sequence are identical to residues determined by Edman degradation from a protein found in gel filtration-separated venom fractions 23–26. Overlined are three histidine– containing regions known to bind copper in the active site of the Coprinus laccase. Indicated by triangles are six cysteine residues, which occur towards the N-terminus of lac1.
N.M. Parkinson et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 513–520
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Fig. 4. Encoded sequence of tre1. The sequence is aligned with the trehalase from the beetle T. molitor, identified by the protein database reference P32359. The signal sequence in tre1 is italicised. Residues immediately following the predicted signal sequence cleavage site, which are identical to a protein found in gel filtration-separated venom fractions 32–35, are doubly underlined. Sequences conserved in trehalases are also present in tre1, and are singly underlined.
pla) trehalose is the major storage carbohydrate (Mullins, 1985). The discovery of a putative venom trehalase, tre1, suggests that the production of haemolymph glucose may be involved in host conditioning. The activity of this enzyme may provide glucose for the developing wasp larva, which would indicate a nutritional function for venom. Our study has provided insights into the complexity and molecular composition of a venom which is a rich source of proteins that antagonise haemocyte functions (Parkinson et al., 2002a).
Several venom proteins that can be considered as candidates for anti-haemocyte activity have been identified. Knowledge of the primary structure of venom proteins will aid their further purification, and facilitate studies that could provide a more detailed understanding of their function and contribution to host conditioning. Acknowledgments This work was supported by the Pesticides Safety Directorate of the Department for Environment, Food and Rural Affairs (DEFRA).
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References Ducros, V., Brzozowski, A.M., Wilson, K.S., Brown, S.H., Ostergaard, P., Schneider, P., Yaver, D.S., Pedersen, A.H., Davies, G.J., 1998. Crystal structure of the type-2 Cu ˚ resolution. depleted laccase from Coprinus cinereus at 2.2 A Nat. Struct. Biol. 4, 310–316. Jones, D., Sawicki, G., Wozniak, M., 1992. Sequence, structure, and expression of a wasp venom protein with a negatively charged signal peptide and a novel repeating internal structure. J. Biol. Chem. 267, 14871–14878. Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C.J., Hofmann, K., Bairoch, A., 2002. The PROSITE database, its status in 2002. Nucleic Acids Res. 30, 235–238. Kitano, H., 1982. Effect of the venom of the gregarious parasitoid Apanteles glomeratus on its hemocyte encapsulation by the host. Pieris. J. Invertebr. Pathol. 40, 61–67. Krishnan, A., Nair, P.N., Jones, D., 1994. Isolation, cloning, and characterization of new chitinase stored in active form in chitin-lined venom reservoir. J. Biol. Chem. 269, 20971–20976. Mullins, D.E., 1985. Chemistry and physiology of the hemolymph. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 3. Pergamon Press, Oxford, pp. 355–400. Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Osman, S.E., 1978. Die Wirkung der Sekrete der weiblichen ¨ Genitalanhangsdrusen von Pimpla turionellae L (Hym., ¨ Ichneumonidae) auf die Haemocyten und die Einkapselungsreaktion von Wirtspuppen. Z. Parasitenkd. 57, 89–100. Parkinson, N., Weaver, R.J., 1999. Noxious components of venom from the pupa-specific parasitoid Pimpla hypochondriaca. J. Invertebr. Pathol. 73, 74–83. Parkinson, N.M., Smith, I., Weaver, R., Edwards, J.P., 2001. A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca. Insect Biochem. Mol. Biol. 31, 57–63. Parkinson, N., Richards, E.H., Conyers, C., Smith I., Edwards, J.P., 2002a. Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein. Insect Biochem. Mol. Biol. 32, 729–735.
Parkinson, N., Conyers, C., Smith, I., 2002b. A venom protein from the endoparasitoid wasp Pimpla hypochondriaca is similar to snake venom reprolysin-type metalloproteases. J. Invertebr. Pathol. 79, 129–131. Richards, E.H., Parkinson, N.M., 2000. Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea. J. Invertebr. Pathol. 73, 74–83. Rotheram, S., 1973. The surface of the egg of a parasitic insect II. The ultrastructure of the particulate coat on the egg of Nemeritus. Proc. Roy. Soc. Lond. (B) 183, 195–204. Salt, G., 1957. Experimental studies in insect parasitism X. The reactions of some endopterygote insects to an alien parasite. Proc. R. Soc. Lond. (B) 147, 167–184. Schmidt, O., Theopold, U., Strand, M.R., 2001. Innate immunity and evasion by insect parasitoids. BioEssays 23, 344–351. Stanley, D.W., Ho, R.W., 2001. Eicosanoids in insect immune signal transduction. In: Beck, G., Sugumaran, M., Cooper, E.L. (Eds.), Phylogenetic Perspectives on the Vertebrate Immune System (Advances in Experimental Medicine, 484). Kluwer Academic Publishers, New York, pp. 265–273. Strand, M.R., Pech, L.L., 1995. Immunological basis for compatibility in parasitoid-host relationships. Annu. Rev. Entomol. 40, 31–56. Takiguchi, M., Niimi, T., Su, Z-H., Yaginuma, T., 1992. Trehalase from male accessory gland of an insect, Tenebrio molitor. Biochem. J. 288, 19–22. Tanaka, T., 1987. Effect of the venom of the endoparasitoid, Apanteles kariyai Watanabe, on the cellular defence reaction of the host, Pseudaletia separata Walker. J. Insect Physiol. 33, 413–420. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Vinson, S.B., Scott, J.R., 1974. Particles containing DNA associated with oocytes of an insect parasitoid. J. Invertebr. Pathol. 25, 375–378. Webb, B.A., Luckhart, S., 1994. Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens. Arch. Insect Biochem. Physiol. 26, 147–163.
cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis Neil M. Parkinsona,*, Christine M. Conyersa, Jeff N. Keenb, Alan D. MacNicolla, Ian Smitha,1, Robert J. Weavera a Plant Health Group, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK School of Biochemistry and Molecular Biology, Astbury Building University of Leeds, Leeds LS2 9JT, UK
b
Received 9 October 2002; received in revised form 29 January 2003; accepted 12 February 2003
Abstract Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro antihaemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large ()30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: cDNA; Pimpla hypochondriaca; Parasitoid; Hymenoptera; Wasp; Venom; Laccase; Trehalase; vpr
1. Introduction Insects possess a potent cellular immune system which is able to encapsulate foreign materials, including parasitoid eggs implanted into the haemocoel of hosts (Salt, 1957; Strand and Pech, 1995; Schmidt et al., 2001; Stanley and Ho, 2001). *Corresponding author. Tel.: 44-1904-462268; fax.: 44-1904-462111. E-mail address: [email protected] (N.M. Parkinson). 1 Present address: Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
At the time of oviposition, adult female parasitoids inject their hosts with various secretions, which are derived from ovarian tissue and venom. Factors produced in the ovarian tract which prevent the host’s immune system from destroying parasitoid eggs include polydnaviruses, virus-like particles and proteins (Vinson and Scott, 1974; Rotheram, 1973; Webb and Luckhart, 1994). Aside from our work, with the pupal endoparasitoid Pimpla hypochondriaca, relatively few studies have described the contribution of parasitoid venom to host immune suppression (Osman,
1532-0456/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 3 . 0 0 0 4 1 - 3
514
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1978; Kitano, 1982; Tanaka, 1987) and still less is known about the nature of venom components, although two cDNAs encoding proteins found in the venom gland of Chelonus sp. have been reported (Jones et al., 1992; Krishnan et al., 1994). We have established that venom from P. hypochondriaca has potent effects against haemocytes maintained in culture, and prevents encapsulation responses when injected into the haemocoel (Richards and Parkinson, 2000; Parkinson et al., 2002a). Previously, we have described paralytic and cytotoxic factors in venom fractionated by gel filtration, in addition to identifying L-DOPA oxidising activity (Parkinson and Weaver, 1999). A cDNA encoding an arthropod-specific phenoloxidase, an enzyme usually found in haemocytes, has been cloned from a cDNA library made from the venom-synthesising gland (Parkinson et al., 2001). Most recently, we have characterised cDNAs encoding a venom serine protease and a reprolysintype protease by random sequence analysis of clones in this library (Parkinson et al., 2002a,b). We have now extended the cDNA library sequence analysis and determined the primary structure of a further five proteins larger than 30 kDa, and additionally, confirmed these as venom constituents by N-terminal sequence analysis. 2. Materials and methods 2.1. Cloning and sequencing of cDNAs Lambda ZAP clones from a previously constructed venom gland library (Parkinson et al., 2001) were converted to p-Bluescript plasmids by in vivo excision according to the supplier’s protocol (Stratagene). Ampicillin-resistant colonies were selected and used to make broth cultures from which phagemid DNA was purified. The 59 end sequence of 500 cDNA clones was obtained using an automated DNA sequencer (ABI Prism 377). cDNAs predicted to encode proteins with signal sequences were identified using the SignalP program (Nielsen et al., 1997; http:yy www.cbs.dtu.dkyservicesysignalP-2.0). Clones encoding sequences, which matched N-terminal sequence from venom protein were fully sequenced for both DNA strands. Sequences were aligned using the CLUSTALW program (Thompson et al., 1994).
2.2. Separation of venom proteins and N-terminal sequencing Venom was size-fractionated using high-resolution gel filtration chromatography as described previously (Parkinson and Weaver, 1999). Pooled gel filtration-separated venom from fractions 23– 26, 30–32, 32–35 and 44–45 was concentrated 10-fold using an ultrafiltration device (UltrafreeMC Millipore, Bedford, MA). Venom proteins were separated by SDS-PAGE using 8, 10 or 12% acrylamide gels, and protein masses were estimated by reference to standards of 97, 66, 45, 21 and 14 kDa (BioRad). Separated proteins were blotted onto PVDF membrane at 30 V for 16 h in Towbin buffer using a tank blotter, as described previously (Parkinson et al., 2002a). N-terminal sequences (at least six residues) of blotted proteins were determined by Edman degradation using an Applied Biosystems Procise 494 HT sequencer. 3. Results 3.1. cDNAs encoding venom proteins greater than 30 kDa Listed in Table 1 are the GenBank accession numbers for five cDNAs encoding venom proteins, all of which possess signal sequences that are highlighted in the full sequences shown in Figs. 1 and 2 Fig. 3 Fig. 4. The predicted post-signal peptide sequences from the cDNAs are indicated, along with the matching sequence determined for venom proteins by Edman degradation. Following blotting and staining, vpr1, vpr2 and lac1 stained well and could easily be related to individual abundant venom proteins reported previously (Parkinson et al., 2002a); vpr2 and tre1 proteins stained less well after blotting (data not shown). Also indicated in Table 1 are the masses of the proteins calculated from the deduced amino acid sequences, the mass estimated from SDS-PAGE gels, and the fractions of gel filtration-separated venom in which the proteins were identified. 3.2. Venom proteins 3.2.1. vpr1 and vpr3 A BLAST search of the GenBank database revealed that the vpr1 sequence shares 63% amino acid identity with the Pimpla venom protein vpr3. Alignment of these two sequences (Fig. 1) with
CDNA
Accession No.
Protein N-terminus
Predicted N-terminus
Calculated Mass Da
SDS-PAGE Mass Da
Gel Filtration Fractions
Homology
lac1 tre1 vpr1 vpr2 vpr3
AJ427356 AJ459958 AJ459813 AJ459811 AJ459812
DNDGIT GSIGHV EGAGFVL DAVNEG DSDIYL
DNDGIT GSIGHV EGAGFVL DAVNEG DSDIYL
74020 63413 32929 53263 32723
76000 61000 32000 57000 32000
23–26 32–35 30–32 30–32 44–45
laccases trehalases vpr3 Drosophila gene vpr1
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Table 1
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Fig. 1. Encoded sequence and alignment of vpr1 and vpr3. Signal sequences are italicised. Residues at the beginning of the mature proteins, which are identical to N-terminal sequence, determined for venom proteins found in gel filtration-separated venom fractions 30–32 (vpr1) and 44–45 (vpr3), are underlined.
ClustalW indicated that the sequences are most dissimilar in the N-terminal one-third of proteins. No other proteins in the GenBank database had close sequence similar to vpr1 or vpr3. Despite having very similar masses, vpr1 and vpr3 eluted in disparate fractions (30–32 and 44–45, respectively) of gel filtration-separated venom. 3.2.2. vpr2 The vpr2 sequence was most similar to a protein identified from the genome sequence of Drosophila melanogaster (accession number AAF46793; 37% amino acid identity), with the greatest sequence similarity occurring in the carboxyl half of the proteins (Fig. 2). The longest contiguous sequence identity was 14 residues (IFKGDLNYRKLLGD; vpr2 residues 399–412). The GenBank database search identified proteins containing this sequence that were derived from genes present in the human (NM_024573), nematode (NM_075624), and yeast (NC_001145) genomes, indicating that genes related to vpr2 occur in a wide range of phyla. No function has been determined for any of these proteins. 3.2.3. lac1 When the predicted sequence of lac1 was used to search GenBank, similarity was detected to laccases from several wood-rotting fungi and to six proteins of unknown function identified from the Drosophila genome sequence. The lac1 sequence was aligned (Fig. 3) with the Coprinus cinereus laccase and with a Drosophila gene prod-
uct (AAF 57331), the latter having the highest degree of sequence identity (46%) to lac1. The crystal structure of the laccase from the ink cap fungus Coprinus cinereus has recently been resolved (Ducros et al., 1998) and copper-binding regions have been identified. Alignment with the fungal laccase indicated that lac1 shares 27% residue identity and contains all three sequences that bind copper in this enzyme. A Cysteine-rich region containing six cysteine residues occurs towards the N-terminus in both lac1 and the Drosophila gene products, but is absent from the Coprinus sequence. 3.2.4. tre1 A BLAST search indicated that tre1 was similar to trehalases from a wide range of organisms, and most similar to a trehalase from the bean shaped accessory gland of the mealworm Tenebrio molitor (Takiguchi et al., 1992). The tre1 and mealworm cDNAs aligned closely (Fig. 4) and shared 49% amino acid identity. Underlined in the Figure are two sequences conserved in trehalases, P-G-G-RF-x-E-x-Y-x-W-D-x-Y and Q-W-D-x-P-x-wGAx-WwPASx-P, identified in the Expasy Prosite entry PDOC00717 (Falquet et al., 2002). 4. Discussion We have demonstrated the utility of random sequence analysis of a cDNA library in providing a rapid analysis of the genes that encode many of the components of a complex venom. This cloning
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Fig. 2. Encoded sequence of vpr2. The sequence is aligned with that of a Drosophila gene product having the protein database identifier CG11474. The signal sequence of vpr2 is italicised. Residues at the beginning of the mature protein, which are identical to the Nterminus of a venom protein found in gel filtration-separated venom fractions 30–32, are underlined.
approach could be more widely applicable to the study of other complex secretory systems where cDNAs encoding proteins of interest are relatively abundant. It is difficult to estimate the diversity of venom components from SDS-PAGE gels, as individual proteins can have variable mobility, depending on the degree of post-translational modification. The current study has confirmed that the complex protein profile seen in SDS-PAGE gels of Pimpla venom is due to the expression of a diverse array of venom-encoding genes. vpr1 and vpr3 both have a very similar signal sequence and shares 63% overall amino acid identity. While the genes encoding these two proteins are related, sequence dissimilarities suggest that they have diverged at an early period in the evolutionary history of P. hypochondriaca. A search of the GenBank database did not reveal any sequences that were closely similar to either vpr1 or vpr3, and these proteins might have become highly specialised. The vpr2 protein is similar to a gene product identified in Drosophila whose function has not yet been determined, and genes, encoding a related protein have been identified in the nematode Cae-
norhabditis elegans and the yeast Saccharomyces cerevisiae. The Drosophila vpr2 homologue does not contain a signal sequence and the venom protein must have been derived from a similar Pimpla gene through an acquisition of sequence encoding a signal peptide. Identification of the venom protein lac1 suggests that the L-DOPA oxidising activity in venom, fractionated by gel filtration (Parkinson and Weaver, 1999) may be mediated both by the previously described arthropod haemocyte-type venom phenoloxidase and by a laccase-type enzyme. In support of this, lac1 is found in gel filtration fractions, previously shown to contain L-DOPA oxidising activity. In this study, the absence of anti-haemocytic activity in gel filtration separated venom containing lac1 reported previously (Parkinson et al., 2002a), may be due to the presence of a phenoloxidase inhibitor. The six-cysteine residues toward the N-terminus of lac1 and the Drosophila homologue may form a distinct structural motif. Pimpla lac1 may be the first insect laccase to be cloned, and further studies are needed to define its activity. In haemolymph of lepidopteran pupae (the developmental stage which is parasitised by Pim-
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Fig. 3. Encoded sequence of lac1. The sequence is aligned with a Drosophila gene and the laccase from the fungus C. cinereus, which have the protein database identifiers AAF 57331 and AAD 30964, respectively. The lac1 signal sequence is italicised and the start of the mature protein is indicated with an arrow. Shaded residues immediately following the signal sequence are identical to residues determined by Edman degradation from a protein found in gel filtration-separated venom fractions 23–26. Overlined are three histidine– containing regions known to bind copper in the active site of the Coprinus laccase. Indicated by triangles are six cysteine residues, which occur towards the N-terminus of lac1.
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Fig. 4. Encoded sequence of tre1. The sequence is aligned with the trehalase from the beetle T. molitor, identified by the protein database reference P32359. The signal sequence in tre1 is italicised. Residues immediately following the predicted signal sequence cleavage site, which are identical to a protein found in gel filtration-separated venom fractions 32–35, are doubly underlined. Sequences conserved in trehalases are also present in tre1, and are singly underlined.
pla) trehalose is the major storage carbohydrate (Mullins, 1985). The discovery of a putative venom trehalase, tre1, suggests that the production of haemolymph glucose may be involved in host conditioning. The activity of this enzyme may provide glucose for the developing wasp larva, which would indicate a nutritional function for venom. Our study has provided insights into the complexity and molecular composition of a venom which is a rich source of proteins that antagonise haemocyte functions (Parkinson et al., 2002a).
Several venom proteins that can be considered as candidates for anti-haemocyte activity have been identified. Knowledge of the primary structure of venom proteins will aid their further purification, and facilitate studies that could provide a more detailed understanding of their function and contribution to host conditioning. Acknowledgments This work was supported by the Pesticides Safety Directorate of the Department for Environment, Food and Rural Affairs (DEFRA).
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