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A new form of arthropod phenoloxidase is abundant in venom of
the parasitoid wasp
Pimpla hypochondriaca
Neil Parkinson
*, Ian Smith, Robert Weaver, John P. Edwards
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK
Received 13 March 2000; received in revised form 22 May 2000; accepted 25 May 2000
Abstract
We have recently identified phenoloxidase (PO) activity among several biologically active factors in venom from the parasitoid wasp Pimpla hypochondriaca. We have now isolated three genes, designated POI, POII and POIII, from a cDNA library made from venom-producing glands and found that their products are related to pro-phenoloxidases (PPOs), which are expressed as proenzymes in haemocytes and which mediate immune processes in arthropods. This is the first report of PO as a venom constituent. Amino acid sequence comparisons between the three PimplaPOs and PPOs revealed several notable differences, including the absence of sequences which specify the site of proteolytic activation in insect PPOs and the unprecedented occurrence of signal peptide sequences. NH2-terminal amino acid analysis of PO purified from venom yielded a peptide sequence matching the predicted
mature NH2termini of POI and POII, confirming the authenticity of the signal peptide and indicating that proteolytic processing,
other than to remove the signal peptide, does not occur in the wasp. Expression of POI, analysed by Northern hybridization, was approximately uniform from the time of adult emergence to day 6 post-emergence, after which it declined. A novel means of host immune suppression, mediated by the unregulated activity of venom PO in the haemocoel, is proposed.2001 Elsevier Science Ltd. All rights reserved.
Keywords: Pimpla hypochondriaca; hymenoptera; parasitoid; venom; phenoloxidase; cDNA
1. Introduction
Parasitoid wasps are important regulators of insect populations and comprise a large proportion of hymen-opteran species (Quicke, 1997). Their eggs are laid on or inside the host, which is subsequently used as a food source by the developing parasitoid larva. Endoparasito-ids, whose eggs are deposited within the haemocoel, must contend with host immune responses that are cap-able of encapsulating and destroying artificially implanted eggs (Feddersen et al., 1986). To circumvent this response the female stings its host, injecting ovarian secretions which contain factors that neutralize host immune function. These host conditioning factors (reviewed by Vinson, 1990; Lavine and Beckage, 1995) include virus-like particles, which mask the parasitoid
* Corresponding author. Tel.: +44-1904-462000; fax: +44-1904-462252.
E-mail address:[email protected] (N. Parkinson).
0965-1748/01/$ - see front matter2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 0 5 - 3
egg from the immune system; polydnaviruses, which infect and disrupt haemocytes; and ovarian proteins, which also target haemocytes (Feddersen et al., 1986; Vinson and Scott, 1974; Webb and Luckhart, 1994).
In addition to ovarian secretions, parasitoids also inject their hosts with venom. The venom of many ecto-parasitoids induces host paralysis, preventing attack of the wasp’s eggs and larvae by biting, and is also involved in the inhibition of moulting (Weaver et al., 1997; Doury et al., 1995; Beard, 1963). In contrast, little is known about the function of endoparasitoid venom, although a role in disabling haemocytes has been sug-gested (Tanaka, 1987; Osman, 1978; Kitano, 1982). Still less is known of the nature of venom components. While two polypeptides occurring in venom of Chelonus sp., an endoparasitoid that oviposits into the egg of its host, have recently been characterized (Jones et al., 1992; Krishnan et al., 1994) the role of these proteins (a chitin-ase and a polypeptide of unknown function) in the para-sitic process is not clear.
endopara-sitoid Pimpla hypochondriaca, and have previously identified several biologically active components includ-ing an enzyme which oxidizes L-DOPA (Parkinson and Weaver, 1999). Two enzymes known to catalyse oxi-dation of L-DOPA are tyrosinase (EC 1.14.18.1 mono-phenol monooxygenase; L-DOPA:oxygen oxidore-ductase) and laccase (EC 1.10.3.1 o-diphenol oxidase; 1,2-benzenediol:oxygen oxidoreductase). Tyrosinases are best known in catalysing synthesis of reactants involved in early steps in the biosynthesis of melanin, a heteropolymer occurring widely in animals which serves both as a pigment and to protect from ultraviolet light (Mason, 1965). Laccases are found in insect integument and catalyse the initial reactions leading to the synthesis of compounds that cross-link integument proteins to chi-tin (Sugumaran, 1988). This process is responsible for tanning the soft, newly synthesized cuticle which is ther-eby converted into a hard and rigid structure.
Tyrosinase activity has been identified in arthropod haemolymph, and cDNAs encoding the enzyme have been cloned from haemocytes of several insect and one crustacean species. Phylogenetic analysis has identified the tyrosinase isolated from arthropod haemocytes as a distinct type (Hall et al., 1995; Kawabata et al., 1995; Fujimoto et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b; Park et al., 1997), which displays sequence similarity to arthropod haemocyanins and is expressed as an inactive proenzyme (Kawabata et al., 1995). The term pro-phenoloxidase (PPO) or, following activation by proteolytic cleavage, phenoloxidase (PO) is com-monly used to distinguish arthropod-specific tyrosinases, and this convention is followed here. PO is closely asso-ciated with the arthropod immune system, and cleavage of PPO to yield PO occurs in response to microbial cell wall products which activate PPO-specific proteases (So¨derha¨ll and Hall, 1984; Ratcliffe and Rowley, 1987; So¨derha¨ll and Smith, 1986; Ashida and Brey, 1997). A mechanism for PO-mediated clearance of microbial infections has recently been proposed, in which quinones generated by PO activity cross-link bacteria to a protein present on the haemocyte cytoplasmic membrane (Marmaras et al., 1996).
We report here the analysis ofPimplavenom fractions containing L-DOPA-oxidizing activity. Genes encoding the proteins in these fractions have been cloned and found to represent a new form of PO, with properties that are strikingly different from those of all other arthro-pod POs which have been characterized to date.
2. Materials and methods
2.1. cDNA library construction
Venom-producing glands from adult female P. hyp-ochondriaca, cultured as described previously
(Parkinson and Weaver, 1999), were dissected from venom sacs and stored at 280°C. Total RNA was pur-ified by selective ethanol precipitation from guanidine thiocyanate using an RNA isolation kit (Stratagene). Polyadenylated RNA was selected using oligo[dT] coated magnetic beads (Promega) and converted to dou-ble stranded cDNA which, after size fractionation, was ligated into the uni-ZAP vector (Stratagene).
2.2. cDNA library screening and sequence analysis
Reverse transcriptase–PCR was used to amplify a por-tion of a putative PO gene from total RNA isolated from venom-producing tissue. Two primers were designed from the conserved motifs HHWHWH (59 -CAY-CAYTGGCAYTGGCA-39) and MG(F/Y)PFD (59 -RTCRAANGGRWANCCCAT-39), which are found in the copper A binding region and towards the COOH ter-minus, respectively, of PPOs (see Fig. 1). These primers were used to amplify a 1.3 kbp product, which was cloned using a T-vector (Promega). Partial sequence analysis of the PCR product confirmed its similarity to known PPOs. Following radiolabelling with [α
-32P]dCTP the PCR product was used as a probe for
cDNA library screening. Sequencing of both DNA strands of selected cDNA clones was carried out by Sequiserve (Vaterstetten, Germany). Multiple sequence alignments were produced using ClustalWWW, a
derivative of the ClustalW program (Thompson et al.,
1994) located at the European Bioinformatics Institute Web pages (http://www2.ebi.ac.uk/clustalw/). A phylo-genetic tree was produced from a ‘guide tree’ generated by ClustalWWW, and displayed using TreeView
(Page, 1996).
2.3. Northern hybridization
For Northern hybridization the oligonucleotide 59 -TTCACTTCTTGGCTATTCCCCACGGGAGTGTTCT TGTGATT-39 was used as a probe. This sequence is derived from one of the cDNAs (POI) cloned in this study and is the complement of the nucleotide sequence encoding amino acid residues 51–64 (NHK...), located towards the NH2-terminal region of POI (Fig. 1). The
Fig. 1. Deduced amino acid sequences ofPimplaPOI, POII and POIII and alignment withManducaPPO andLimulushaemocyanin. The PPO sequences used in the alignment are listed in Fig. 2, though only the moth sequence (M.S.PPO) (Hall et al., 1995) is shown. Residues highlighted with arrowheads are conserved in all PPO sequences and in Limulus haemocyanin (L.P.Hcn) but not in POs I–III. The RF residues indicated by a single overline constitute the proteolytic cleavage site in insect PPOs. Conserved histidine-containing motifs involved in copper binding are underlined.
2.4. PO purification, NH2-terminal sequencing, PAGE
analysis and determination of enzyme specific activity
Venom was size-fractionated as described previously (Parkinson and Weaver, 1999). Fractions 17–20, which contained maximum PO activity, were analysed by SDS–PAGE (Laemmli, 1970) without the addition of reducing agent and compared to the peptide profile from non-fractionated venom. These fractions were also used for determination of the NH2-terminal PO sequence, by
Edman degradation, as well as for determination of enzyme specific activity. The latter was done spectro-photometrically using a wavelength of 492 nm with 15 mM L-DOPA as substrate in 10 mM cacodylate, 10 mM calcium chloride buffer pH 6.9.
3. Results
3.1. Cloning, cDNA sequence analysis and gene expression
Approximately 2% of clones in the cDNA library hybridized to the PCR-generated probe. Three clones (POI, POII and POIII) were selected, according to insert size and restriction profile, and sequenced. The POI open reading frame encoded a polypeptide of 699 amino acids
(Mr79,499), POII a polypeptide of 690 amino acids (Mr
79,020), and POIII a polypeptide of 708 amino acids (Mr79,333).
POI shares 77% amino acid identity with POII and 60% amino acid identity with POIII. Fig. 1 shows deduced amino acid sequences of POI–POIII, and their alignment with a PPO and a haemocyanin cloned from the mothManduca sextaand the horseshoe crabLimulus polyphemus, respectively (Hall et al., 1995; Nakashima et al., 1986). The alignment was made using seven PPO genes (Hall et al., 1995; Kawabata et al., 1995; Fujimoto et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b; Park et al., 1997), though only the Manduca sequence is shown. Conserved residues encoded by all the PPOs used to produce the alignment are highlighted on the
Manducasequence in Fig. 1, as are eight conserved resi-dues (arrowheads) which are absent in the deduced sequences of POI, POII or POIII. A search of the Gen-Bank database using BLASTP indicated that polypep-tides encoded by POI, POII and POIII were most similar to PPOs (approximately 48% identity), with lower simi-larity to haemocyanins (up to 40% identity).
Interestingly, POs I–III each encoded a similar NH2
using the SignalP program (http://www.cbs.dtu.dk/ services/signalP), which predicted signal sequences for all three enzymes with potential cleavage sites located between the conserved glycine and aspartate residues (positions 19 and 20, Fig. 1).
A phylogram indicating the degree of relatedness of POI, POII and POIII to previously reported PPOs and to Limulushaemocyanin is shown in Fig. 2. PPOs from flies and moths clustered into two distinct groups, the moth group being divided into two subgroups each con-taining one of two PPO genes cloned from each species. A third major grouping comprised POs I–III and, as a subgroup, the crayfish PPO and Limulus haemocyanin.
Northern hybridization (Fig. 3) using the POI-specific oligonucleotide indicated a transcript length for this gene of approximately 2.4 kb, similar to the size of the POI cDNA clone (2.2 kb). POI was expressed at a similar level from the time of adult emergence through to the third sampling time at 6 days post-emergence, thereafter declining to relatively low levels by day 9.
3.2. SDS–PAGE analysis of purified venom PO, specific activity and determination of NH2-terminal
amino acid sequence
SDS–PAGE analysis of fractionated venom separated by size exclusion chromatography and containing
Fig. 2. Phylogenetic analysis of PO encoded by POI, POII and POIII and relationship to PPOs from the lepidopterans Hyphantria cunea
(H.C. PPO 1, 2),Manduca sexta(M.S. PPO 1, 2) andBombyx mori
(B.M. PPO 1, 2); the dipteransDrosophila melanogaster(D.M. PPO A1) andAnopheles gambiae(A.G. PPO 1, 2); the crayfishPacifasticus leniusculus (P.L. PPO); and to a haemocyanin from Limulus poly-phemus(L.P. Hcn) (Hall et al., 1995; Kawabata et al., 1995; Fujimoto et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b; Park et al., 1997; Nakashima et al., 1986).
Fig. 3. POI gene expression analysis. Total RNA (4µg) isolated from venom-producing tissues dissected from insects at days 0, 1, 3, 6, and 9 post-emergence was hybridized using a POI-specific oligonucleotide as described in the text. The inset (black background) shows the same RNA samples prior to blotting, stained with ethidium bromide to vis-ualize rRNA and indicate RNA loading. The positions of RNA size standards (Life Technologies) are indicated.
maximum PO activity is shown in Fig. 4. Two polypep-tides of similar electrophoretic mobility were detected with Mrs of approximately 80,000, in good agreement
with the Mrs deduced from the cDNA coding region
sequences of POs I–III. These polypeptides co-migrated with two major constituents of whole venom (Fig. 4, lane W), indicating that PO is abundant in thePimpla venom-producing gland. The specific activity of purified PO, assayed using 15 mM L-DOPA, was DA490
8.4/min/mg protein.
The NH2-terminal amino acid sequence of PO purified
from Pimpla venom was determined to be DEXDRDINQEILDQ, which, apart from the ambiguous third residue, is identical to residues 20–33 of POI and POII (Fig. 1). This sequence occurs immediately after the predicted signal peptide sequence, which is thus con-firmed as authentic. Purified venom PO submitted for sequencing contained two polypeptides in approximately equal proportions (Fig. 4, lane P). Apart from a trace sequence, attributed to small quantities of a contaminat-ing peptide, no other sequence was detectable, indicatcontaminat-ing that both polypeptides in the purified venom shared an identical NH2-terminal amino acid sequence which is
present in both POI and POII. In contrast, the polypep-tide encoded by POIII, which has a similar but distinct sequence at the putative mature NH2 terminus, was not
present in detectable quantities in the venom sample used for sequence analysis.
4. Discussion
Alignment of the deduced amino acid sequences of PPOs with those of PimplaPOs I–III (Fig. 1) indicates extensive similarity, and these genes are clearly related. The Pimpla venom L-DOPA oxidizing activity pre-viously identified in venom fractions (Parkinson and Weaver, 1999) is thus unambiguously attributed to the arthropod-specific PO enzyme type. PPO cDNAs cloned previously are probably derived exclusively from hae-mocytes and expression of POs I–III in secretory epider-mis, the site of venom production, represents a novel source and biological context for the enzyme. Gene expression analysis of maturing venom glands using the POI-specific probe (Fig. 3) revealed a similar level of expression through the first week after adult emergence, followed by a decline to relatively low levels observed in glands 9 days after emergence. The reduction of tran-scription at this time may coincide with a shut-off of venom production as the venom sac becomes full and indicates that venom secretion may be regulated. Further analysis will be required to establish whether transcrip-tion of PO genes is restored in response to venom depletion during oviposition.
Pimpla POs I–III are closely related to one another, and it is most likely that their genes have evolved by duplications which have occurred at different times; the high degree of similarity between POI and POII indi-cates a recent duplication. The increase in Pimpla PO gene number could have evolved as a means of maxim-izing PO production in the venom-producing glands.
There are several notable differences in primary struc-ture between the polypeptides encoded by POs I–III and those of previously cloned PPO genes. Most striking is the presence in all three Pimpla venom POs of highly-conserved signal sequences, which direct polypeptides into the endoplasmic reticulum to initiate secretion (Leader, 1979), and this is the first report that PO can be secreted using the endoplasmic reticulum system. In contrast, PPOs isolated from haemocytes have not been shown to contain recognised signal peptides and are released by cell rupture (Jiang et al., 1997b; So¨derha¨ll and Smith, 1986), a process involving cell destruction and which would thus be inappropriate for venom-pro-ducing cells. Recently, a protein homologous to arthro-pod haemocyanins, and which contains a signal peptide sequence, has been identified in embryos of the grass-hopperSchistocerca americana (Sanchez et al., 1998).
PO fromPimplavenom is active without the addition of exogenous proteases, and suppression of enzyme activity during storage in the venom sac appears to be mediated by heat-stable inhibitors present in venom (Parkinson and Weaver, 1999). The NH2-terminal
sequence analysis of venom PO confirms that proteolytic processing of POI and POII, other than removal of the signal peptides, does not occur in Pimpla. The absence of proteolytic cleavage in venom PO suggests that the enzyme is constitutively active, which is consistent with the proposed role of venom PO inhibitors in preventing PO activity whilst it is stored in the venom sac. Additionally, the highly conserved RF motif, which serves as the proteolytic cleavage site in all insect PPOs (Fig. 1), is also absent in POs I–III, consistent with a lack of a requirement for proteolytic activation. We can-not, however, rule out the possibility that the observed venom PO activity is due to a low level of proteolytic processing, which might have generated processed enzyme at a concentration below that detectable in the NH2-terminal sequence analysis, and further studies will
be required to clarify this issue.
Pimpla venom PO elutes from gel filtration columns as a broad peak (Parkinson and Weaver, 1999), suggest-ing a wide mass range for the enzyme. Analysis of PPO from Manduca sexta has indicated that this enzyme can occur in a variety of multimeric forms (Jiang et al., 1997b), and similar structural forms may also exist for
Pimpla venom PO which would account for its wide mass range. The absence of a detectable NH2-terminal
amino acid sequence corresponding to POIII may be explained if this polypeptide has a mobility different to that of the majority of PO: as only those fractions with maximal PO activity were submitted for NH2-terminal
sequencing, the polypeptide encoded by POIII may have been excluded from the fractions sent for NH2-terminal
sequence analysis.
et al., 1995; Burmester and Scheller, 1996), and the phy-logram shown in Fig. 2 indicates that venom PO genes cluster in a group which also contains the crayfish PPO andLimulushaemocyanin. Crayfish PPO and the haemo-cyanin ‘a’ from Eurypelma californicum andPanulirus interruptushave previously been shown to share exten-sive amino acid identity, which has led to the recent suggestion that haemocyanins are derived from POs or PO-like enzymes (Burmester and Scheller, 1996). The extensive amino acid sequence identity which exists between the Pimpla venom POs and Limulus haemo-cyanin (Fig. 1) confirms the close relationship between arthropod phenoloxidases and haemocyanins.
SDS–PAGE analysis ofPimpla venom (Fig. 4) indi-cates that PO is a major secretory product, which is con-sistent with the high enzyme activity previously reported for this fluid (Parkinson and Weaver, 1999). Parasitoid venoms are substantially diluted when they are injected into the host, and the abundance of PO inPimplavenom suggests that it may play a role in host conditioning.
Whilst enzymes are commonly found as venom components, this is the first report of PO as a venom constituent and invites speculation as to its function. Although the mode of action of PO products generated from the enzyme derived from haemocytes is not fully understood, recent studies have identified a protein located in haemocyte plasma membranes which partici-pates, with PO, in a process that entraps bacteria (Marmaras et al., 1996). This provides evidence that PO products can bind to the haemocyte plasma membrane. Damage to haemocytes, including lysis and degranu-lation, has also been attributed to their over-stimulation by PO products (Smith and So¨derha¨ll, 1983). Venom PO derived products, if produced in sufficient quantity in vivo, could similarly bind to haemocytes and disrupt their function, thus contributing to host immune sup-pression. Whilst the precise effects ofPimplavenom PO remain to be established in vivo, we propose a novel mode of host immune suppression mediated through the unregulated synthesis of PO products which impair haemocyte function.
Acknowledgements
We thank J. Keen, University of Leeds and A. Moire´, University of Sheffield for peptide sequencing. This work was supported by the Pesticides Safety Directorate of the Ministry of Agriculture, Fisheries and Food.
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