Phospholipid-hydroperoxide glutathione peroxide (PHGPx) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln82 and Trp134. Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H2O2 using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H2O2 exposure, external temperature shock, and starvation. Under H2O2 overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H2O2.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that loss of expression of BiPrx1 or BiTPx1 is compensated by up-regulation of expression of the other peroxidase in response to H2O2 overload.

A thioredoxin peroxidase (TPx) gene was cloned from the bumblebee, Bumbus ignitus. The B. ignitus TPx (BiTPx) contains an open reading frame of 585 bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BiTPx cDNA showed 90% identity to Apis melifera (AmTPx-1), 80% to Aedes aegypti (AaTPx), and 78% - 47% to other insect 2-Cys TPx. Northern blot analysis revealed the presence of BiTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BiTPx in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting the BiTPx is not secretable. The cDNA encoding BiTPx was expressed as a 27-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BiTPx was shown to reduce H2O2 in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor.