The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat.
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.
Montesino
Raquel
R
Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Gil
Jeovanis
J
González
Luis J
LJ
Zamora
Yasser
Y
Royle
Louise
L
Rudd
Pauline M
PM
Dwek
Raymond A
RA
Harvey
David J
DJ
Cremata
José A
JA
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
D000956:Antigens, Viral; D011134:Polysaccharides; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C087086:glycoprotein E2, classical swine fever virus