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The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat |
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| Authors: | Raquel Montesino Luis J González Louise Royle Pauline M Rudd David J Harvey |
| Institution: | a Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba b Department of Proteomics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba c National Institute for Bioprocessing Research and Training, Dublin-Oxford Glycobiology Laboratory, Conway Institute, University College Dublin, Dublin 4, Ireland d Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK |
| Abstract: | Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man8GlcNAc2 isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk. |
| Keywords: | Classical swine fever virus E2 glycoprotein Goat milk N-glycosylation pattern |
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