Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A2 (PLA2) is one of the main components of bee venom. Here, we identify a venom PLA2 from the bumblebee, Bombus ignitus. Bumblebee venom PLA2 (Bi-PLA2) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA2 gene. Bi-PLA2 is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA2 (136 amino acids) possesses features consistent with other bee PLA2s, including ten conserved cysteine residues, as well as a highly conserved Ca2+-binding site and active site. Phylogenetic analysis of bee PLA2s separated the bumblebee and honeybee PLA2 proteins into two groups. The mature Bi-PLA2 purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA2. Immunofluorescence staining of Bi-PLA2-treated insect Sf9 cells revealed that Bi-PLA2 binds at the cell membrane and induces apoptotic cell death.