Project description:Ebola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. The glycoprotein possesses a large mucin-like domain responsible for the cytopathic effect on infected cells, yet its structure or potential role in early entry events is poorly defined. To understand the importance of O-glycans and the individual O-glycosylation sites for viral infectivity, we performed a comprehensive characterization of site-specific glycosylation governed by the three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, initiating O-glycan biosynthesis. Using TMT isobaric labelling we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for each of the three GalNAc-Ts, as well as compared it to patterns on wild type virus-like particles. In total we found 38 and 41 O-glycosites on virus like particle-derived and recombinant GP, respectively, with well correlated sites and site-specific structures. Examination of the isoform-specific glycosylation demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. We next demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. This work represents a comprehensive site-specific analysis of EBOV GP and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.
Project description:Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal main- tenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determina- tion of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.