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 poly protein 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-E2 E1-E2 d E2 form the associated E1-E2 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