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

Soldatova, Lyudmila N. and Tsai, Chaoming and Dobrovolskaia, Ekaterina and Marković-Housley, Zora and Slater, Jay E.. (2007) Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells. Allergy and asthma proceedings, Vol. 28, no. 2. pp. 210-215.

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Official URL: http://edoc.unibas.ch/dok/A5262590

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Abstract

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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Schirmer)
UniBasel Contributors:Schirmer, Tilman and Housley-Markovic, Zora
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:OceanSide Publications
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:04 Sep 2015 14:31
Deposited On:22 Mar 2012 13:51

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