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We report the cloning of a gene and the characterization of the encoded protein, which is released by the teratocytes of the parasitoid Aphidius ervi in the haemocoel of the host aphid Acyrthosiphon pisum. The studied protein was identified by LC-MS/MS, and the gathered information used for isolating the full length cDNA. The corresponding gene was made of 3 exons and 2 introns, and was highly expressed in the adult wasps and in parasitized hosts. The translation product, which was named Ae-ENO, showed a very high level of sequence identity with insect enolases. In vivo immunodetection experiments evidenced Ae-ENO localization in round spots, present in the teratocytes and released in the host haemocoel. Moreover, strong immunoreactivity was detected on the surface of A. ervi larvae and of host embryos. Ae-ENO expressed in insect cells was not secreted in the medium, indicating the occurrence in the teratocytes of an unknown pathway for Ae-ENO release. The recombinant protein produced in bacteria under native conditions was a dimer, with evident enolase activity (K(m) = 0.086 +/- 0.017 mM). Enolase is a well known enzyme in cell metabolism, which, however, is associated with a multifunctional role in disease, when present in the extracellular environment, on the surface of prokaryotic and eukaryotic cells. In these cases, the enolase mediates the activation of enzymes involved in the invasion of tissues by pathogens and tumour cells, and in the evasion of host immune response. The possible role played by Ae-ENO in the host regulation process is discussed in the light of this information.

Parasitism by the endophagous braconid Aphidius ervi (Hymenoptera, Braconidae) has a negative impact on the reproductive activity of its host, Acyrthosiphon pisum (Homoptera, Aphididae). The host castration is induced by the parasitoid venom and is reproduced by the injection of chromatographic fractions highly enriched with two proteins, of 18 (p18) and 36 kDa (p36) in size, respectively. Here we demonstrate that these bioactive proteins trigger apoptosis of the cells in the germaria and ovariole sheath of the host aphid. Both p18 and p36 were internally sequenced and the gathered information was matched against the deduced amino acid sequence of the putative proteins encoded by cDNA clones, randomly selected from a cDNA library, which was raised using mRNA extracted from A. ervi venom glands. The identified cDNA clones contained an insert corresponding to the RNA product of an interrupted gene, made of six exons and five introns, which was found to be transcribed at higher levels in adult females of A. ervi than in males. This gene codes for a putative protein composed of 541 amino acids, with a calculated molecular mass of 56.9 kDa, which contained the amino acid sequences experimentally determined for both p18 and p36. This putative protein showed a significant level of sequence identity with gamma-glutamyl transpeptidases (gamma-GT), and it was named Ae-gamma-GT. The gamma-GTs are enzymes which play a key role in the metabolism of glutathione (GSH) and, as observed in most organisms, they are membrane-bound heterodimers formed by a large and a small subunit, which originate by post-translational processing of a single-chain precursor. The expression in insect cells of Ae-gamma-GT confirmed the occurrence of the expected post-translational processing, and demonstrated that, unlike other gamma-GTs, this protein is secreted in the extracellular environment. A measurable gamma-GT activity was detected in the venom of A. ervi and in the chromatographic fractions containing Ae-gamma-GT. Thus, we suggest that this venom protein may induce apoptosis in the host ovarioles by generating an alteration of the GSH metabolism and a consequent oxidative stress.