CHARACTERIZATION OF THE ISOFORMS OF PHOSPHOLIPASE-A(2) FROM HONEYBEE VENOM

Phospholipase A2 from the venom of the European honeybee (Apis mellifera) consists of three isoforms with approximate molecular masses of 16, 18, and 20 kDa, respectively, as deduced from SDS-PAGE. These variants, termed PLA-16, PLA-18, and PLA-20, were isolated by lectin affinity chromatography and preparative polyacrylamide gel electrophoresis. The amino acid sequences of the N-terminal peptide portions of all three isoforms, as assessed by automated Edman degradation, were identical with that expected for honeybee phospholipase A2. Sequencing data suggest that, while PLA-18 and PLA-20 carry oligosaccharide residues at asparagine-13, PLA-16 has escaped glycosylation during biosynthesis. Release of the carbohydrate from PLA-18 and PLA-20 with peptide:N-glycosidase F abolished the molecular mass differences between the three isoforms of phospholipase. Differences in sensitivity to alpha-mannosidase and monosaccharide composition of PLA-18 and PLA-20 further indicate that their electrophoretic separation is based on structural features of the N-glycosidically linked oligosaccharide. Noticeably, PLA-20 contains N-acetylgalactosamine, a sugar not having yet been described as a constituent of insect glycoproteins.