cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Abstract

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic 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.

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). 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, 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 reprolysin-type protease by random sequence analysis of clones in this library (Parkinson et al., 2002a, Parkinson et al., 2002b).

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.