Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum

Abstract

Using chromogenic substrates 5-bromo-4-chloro-3'-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperature dependent with bivalent cation effects. The full-length cDNA sequence of ALPase was amplified from the cDNA library of the venom apparatus of P. puparum, providing the first molecular characterization of ALPase in the venom of a parasitoid wasp. The cDNA consisted of 2645 bp with a 1623 bp open reading frame coding for 541 deduced amino acids with a predicted molecular mass of 59.83 kDa and pI of 6.98. Using multiple sequence alignment, the deduced amino acid sequence shared high identity to its counterparts from other insects. A signal peptide and a long conserved ALPase gene family signature sequence were observed. The amino acid sequence of this venom protein was characterized with different potential glycosylation, myristoylation, phosphorylation sites and metal ligand sites. The transcript of the ALPase gene was detected by RT-PCR in the venom apparatus with development related expression after adult wasp emergence, suggesting a possible correlation with the oviposition process.

Figure 1:

Localization of ALPase activity in the venom apparatus of Pteromalus puparum by BCIP/NBT staining. (A) ALPase activity in the venom gland; (B) ALPase activity in the venom reservoir; (C and D) Control venom gland and venom reservoir subjected to ALPase staining protocol without chromogenic substrate. High quality figures are available online.

Figure 2:

Ultrastructural localization of ALPase in Pteromalus puparum venom gland secretory cells. Black lead deposits showing the presence of ALPase. (A–B) Observe the presence of this enzyme inside nuclei (Nu) and secretory vesicles (Sv). (C–D) Negative controls. High quality figures are available online.

Figure 3:

Biochemical characterization of venom ALPase of Pteromalus puparum. (A) Thermal stability of venom ALPase of P. puparum. The residual activity of the enzyme was determined at pH 8.5 following heating for 0–30 min. (B) Effect of divalent cations (Mg, Ca, Zn and Mn) on ALPase activity in the venom apparatus of P. puparum. Values are the mean of three independent assays, and error bars are ± SD of the mean. High quality figures are available online.

Figure 4:

Multiple alignment of the deduced amino acid of venom ALPase amino acid sequence from Pteromalus puparum with the counterparts from other species. The sequences used in the alignment are Nasonia vitripennis (accession number XP_001603241 ), Apis mellifera (accession number XP_624078), Tribolium castaneum (accession number XP_9750S0), Aedes aegypti (accession number XP_001657478) and Drosophila melanogaster (accession number NP_524601). Identical amino acids are shaded in black, and similar amino acids are in grey. High quality figures are available online.

Figure 5:

Time-course expression profiles of ALPase in the venom apparatus of Pteromalus puparum from 0 to 7 days after emergence. (A) Agarose gels of RT-PCR amplicons for ALPase and 18S at various times post emergence from one of the three experiments. (B) Enzyme activity profiles of ALPase in the venom apparatus at various times post emergence. Values are the mean of three independent assays, and error bars are ± SD of the mean. High quality figures are available online.