Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Abstract

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co-and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined. (J

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a Broadly Neutralizing Human Antibody

“…Extra side chain rotamers were included to ensure sufficient sampling (-ex1 -ex2 -ex3), and a separate simulation for non-alanine mutants was performed incorporating backbone and off-rotamer side chain minimization of the complex before and after mutation (-min_interface -int_bb -int_chi). Glycans were modeled using the GlyProt web server (48), using oligomannose glycans because high mannose glycans were found to be predominant in native E2 (49). As with the Rosetta simulations, the engineered N-terminal arginine residue was removed from the peptide in the complex structure prior to input into the server.…”

HIV-1 Envelope Glycan Moieties Modulate HIV-1 Transmission

“…However, some glycoproteins, including HIV-1 gp120, demonstrate protein-specific glycosylation (22)(23)(24)(25). Consequently, the glycan profile of HIV-1 is divergent from the normal glycosylation of its host cell.…”

Site Specific Identification of N-Linked Glycosylation in Proteins by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

“…HCV E2 protein expressed in Chinese hamster ovary (CHO) cells was reduced, alkylated and digested with either trypsin or chymotrypsin following the procedure described in [18].…”

“…Recently, we reported the mass spectrometric characterization of the N-glycans attached on the Hepatitis C virus (HCV) E2 glycoprotein [18] (DOI: 10.1016/ j.jasms.2007.11.022). Hepatitis C viral infections represent the main cause of liver diseases in humans, including chronic hepatitis, cirrhosis or hepatocellular carcinoma.…”

“…Using a combination of proteases (trypsin and -chymotrypsin) and nano-scale liquid chromatography in combination with collision induced dissociation (CID) tandem MS, it was possible to characterize the glycoforms associated with each of the 11 glycosylation sites. The major glycans at 9 of the 11 sites are exclusively of high mannose type, comprising species ranging from the minimal core structure Man 3 GlcNAc 2 (Man3) up to 12 hexose residues elongating the chitobiose core (Hex 3 Man 9 GlcNAc 2 ), whereas two sites, N41 (N423) and N48 (N430) proved to be occupied with complex type oligosaccharides of the following sugar composition: Man3-GlcNAc and Man3-GlcNAcFuc at the site N41 and Man3-Fuc, Man3-GlcNAcFuc, Man3-GlcNAc 2 Fuc at the site N48 [18]. Digestion by -chymotrypsin led to formation of glycopeptides of different amino acid compositions containing the same glycosylation site, whose identity was confirmed by tandem MS.…”