Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Abstract

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/ Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5.

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

“…MS/MS data of HBV generated in prior studies (Peiren et al, 2005;Van Vaerenbergh et al, 2013 were reanalyzed by searching against the honeybee protein RefSeq database extended with all translated Api m 10 isoform sequences to identify isoformspecific tryptic peptides. Identical search parameters were used as those previously described (Peiren et al, 2005;Van Vaerenbergh et al, 2013.…”

“…Identical search parameters were used as those previously described (Peiren et al, 2005;Van Vaerenbergh et al, 2013.…”

“…All other Api m 10 variants and uterocalin were cloned, sequenced and expressed using the pET system (Life technologies, Carlsbad, CA, USA) (de Graaf et al, 2010). They were overproduced in Escherichia coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads (adapted protocol from (Van Vaerenbergh et al, 2013). When native purification yielded insufficient amounts of soluble protein, denaturing purifications were applied.…”

“…Purified proteins were dialyzed against phosphate buffered saline (PBS) and sample purity was evaluated by Coomassie staining of SDS-PAGE gels. Protein integrity was verified by Western blots using an antiHis antibody (Van Vaerenbergh et al, 2013).…”

An LCMS method for the assay of melittin in cosmetic formulations containing bee venom

“…Melittin (MW~2,800) is the main constituent of the venom, constituting approximately 45-60 % of the bulk venom material and is a 26-amino acid peptide [4]. The other components are the peptides apamine, mast cell degranulating peptide (MCDP), secapin, adolapin and apidaecin [3,5,6]. Both phospholipase (api m1) and hyaluronidase (api m3) are classified as major allergens according to the International Union of Immunological Societies (IUIS) [7].…”