title: Mass spectrometric analysis of hepatitis C viral envelope protein E2 reveals extended microheterogeneity of mucin-type O-linked glycosylation.
abstract: The infectious liver disease hepatitis C is caused by the small, enveloped, positive single-strand RNA hepatitis C virus (HCV). The HCV genome encodes for a single polyprotein precursor of ∼3010 amino acid residues. Host and cellular proteases co- and posttranslational process the precursor creating six nonstructural (NS) proteins and four structural components. Properly folded forms of the envelope proteins E1 and E2 form the associated E1-E2 complex. This complex represents a significant antigenic component at the viral surface that can interact with several target cell receptors. Extent and type of glycosylation is an important factor for virulence and escape from the immune system. Detailed characterization of the glycosylated sites is helpful for the understanding of different phenotypes as well as for the development of E1/E2-related treatments of HCV infection. Here, we have investigated in detail the O-linked glycosylation of the HCV envelope protein E2 expressed in and isolated from human embryonic kidney (HEK 293) cells. Using nano-liquid chromatography and tandem mass spectrometry approaches, we clearly have identified six residues for O-linked glycosylation within isolated glycopeptides (Ser393, Thr396, Ser401, Ser404, Thr473 and Thr518), carrying mainly Core 1 and Core 2 mucin-type structures. Based on our data, Thr385 is probably glycosylated as well. In addition, we could show that Ser479 within the hyper variable region (HVR) I is not O-glycosylated. For most of these sites, different degrees of microheterogeneity could be verified. Concerning HCV E2, this is the first case of experimentally proven O-linked glycosylation in detail via mass spectrometry.
authors.affiliation: Department of Structural Biology, Centre for Biochemistry and Molecular Biology, Christian-Albrechts Universität, 24118 Kiel, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Bräutigam
authors.forename: Axel J
authors.initials: AJ
authors.lastname: Scheidig
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
mesh_terms: D000595:Amino Acid Sequence; D006031:Glycosylation; D057809:HEK293 Cells; D016174:Hepacivirus / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009077:Mucins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus