title: The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat.
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 Man(8)GlcNAc(2) 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.
authors.affiliation: Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: Raquel
authors.initials: R
authors.lastname: Montesino
authors.forename: Jeovanis
authors.initials: J
authors.lastname: Gil
authors.forename: Luis J
authors.initials: LJ
authors.lastname: González
authors.forename: Yasser
authors.initials: Y
authors.lastname: Zamora
authors.forename: Louise
authors.initials: L
authors.lastname: Royle
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: José A
authors.initials: JA
authors.lastname: Cremata
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D006041:Goats; D008892:Milk / Q000276:immunology* / Q000821:virology*; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D011134:Polysaccharides; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C087086:glycoprotein E2, classical swine fever virus