Scientific Publications by FDA Staff

Allergy Asthma Proc 2007 Mar-Apr;28(2):210-5

Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells.

Soldatova LN, Tsai C, Dobrovolskaia E, Markovic-Housley Z, Slater JE

Slater JE (reprint author), US FDA, Lab Immunobiochem, Ctr Biol Evaluat & Res, 1401 Rockville Pike,HFM 422, Rockville, MD 20852 USA US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20014 USA Univ Basel, Dept Struct Biol, Biozentrum, CH-4003 Basel, Switzerland

Honeybee venom hyaluronidase (Api m 2) is a major glycoprotein allergen. Previous studies have indicated that recombinant Api m 2 expressed in insect cells has enzyme activity and IgE binding comparable with that of native Api m 2. In contrast, Api m 2 expressed in Escherichia coli does not. In this study, we characterized the carbohydrate side chains of Api m 2 expressed in insect cells, and compared our data with the established carbohydrate structure of native Api m 2. We assessed both the monosaccharide and the oligosaccharide content of recombinant Api m 2 using fluorophore-assisted carbohydrate electrophoresis and HPLC. To identify the amino acid residues at which glycosylation occurs, we digested recombinant Api m 2 with endoproteinase Glu-C and identified the fragments that contained carbohydrate by specific staining. Recombinant Api m 2 expressed in insect cells contains N-acetylglucosamine, mannose, and fucose, as well as trace amounts of glucose and galactose, and the oligosaccharide analysis is consistent with heterogeneous oligosaccharide chains consisting of two to seven monosaccharides. No sialic acid or N-acetylgalactosamine were detected. These results are similar to published data for native Api m 2, although some monosaccharide components appear to be absent in the recombinant protein. Analysis of proteolytic digests indicates that of the four candidate N-glycosylation sites, carbohydrate chains are attached at asparagines 115 and 263. Recombinant Api m 2 expressed in insect cells has enzymic activity and IgE binding comparable with the native protein, and its carbohydrate composition is very similar.