Project description:The envelope glycoprotein GP of the ebolaviruses is essential for host cell attachment and entry. It is also the primary target of the protective and neutralizing antibody response in both natural infection and vaccination. GP is heavily glycosylated with up to 17 predicted N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation of GP is important for host cell attachment to cell-surface lectins, as well as GP stability and fusion activity. Moreover, it has been shown to shield GP from neutralizing activity of serum antibodies. Here, we use mass spectrometry-based glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP, including N-, O-, and C-linked glycans.
Project description:Recombinant soluble ectodomains of a prefusion-stabilized MeV F-protein were analysed by bottom-up proteomics to profile the N-glycans.
Project description:The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. Despite significant progress, challenges persist in identifying conserved epitopes capable of inducing broadly protective immune responses, understanding the correlates of protection against influenza infection, and ensuring vaccine safety and efficacy across diverse populations. In this study, we explore the influence of glycan evolution on H3 HA from 1968 to the present. Our in-depth analysis unveiled how glycan addition significantly and differentially impacted HA antigenicity and immunogenicity over time. We observe that adding glycans influenced the polyclonal immune response elicited post-immunization. We propose that the appearance of PNGS at N158 potentially impacted the binding of head-specific broadly neutralizing antibodies (bnAbs). We further observe that, while enzymatic de-glycosylation barely impacted HK/68 HA stability, it destabilized and unfolded in the hyperglycosylated head domain and the membrane-proximal region of the stem domain in Sing/16. These insights expand our understanding of glycans beyond their conventional role in protein folding and as immune evasion tactics and highlight the intricate interplay among glycan integration, viral fitness, and immune recognition. Our findings have implications for vaccine design and manufacturing processes, offering valuable insights for developing more effective influenza vaccines. Moreover, they contribute to ongoing efforts to refine influenza research strategies, emphasizing the potential of stem-directed antibodies in creating more effective and universally applicable influenza vaccines.
Project description:Presentation of self- and foreign peptide antigens by human leukocyte antigen (HLA) complexes at the cell surface is a key process in our immune response. The alpha-chain, the part of the HLA class I complex that contains the peptide binding groove, is one of the most polymorphic proteins in the human proteome. All HLA class I alpha-chains carry a conserved N-glycosylation site, but little is known about its nature and function. Here, we report an in-depth characterization of the N-glycosylation features in HLA class I molecules. In three cell lines we observe that different HLA-A alpha-chains carry similar glycosylation, distinctly different from the HLA-B, HLA-C and HLA-F alpha-chains. HLA-B alpha-chains carry mostly mature glycans, HLA-C and HLA-F alpha-chains carry predominantly high-mannose, whereas HLA-A molecules display the broadest variety of glycan characteristics. We hypothesized that these glycosylation features are directly linked to the cellular localization of the HLA complexes. Analyzing HLA class I complexes from plasma and inner membrane enriched fractions revealed confirmed that most HLA-B complexes can be found in the plasma membrane, most HLA-C and HLA-F molecules reside in the ER and Golgi membrane and HLA-A molecules are more equally distributed over all these cellular compartments. As peptide-binding and specificity is cellular compartment dependent, we corroborate from our data that standard measurements of HLA peptide-antigens from whole cell extracts likely do not exclusively capture the antigen repertoires presented at the cell surface, but also those still within the cell. Our data indicate that standard protein quantification of HLA alpha-chains does not correlate with cell surface expression levels, while analysis of glycopeptides provides allotype and compartment specific quantification.
Project description:Comparison of the glycosylation profile of monoclonal anti-SARS-CoV-2 Spike IgG (87G7) produced in human or fungal expression platforms
Project description:Fusion proteins of the SARS-CoV-1 and SARS-CoV-2 spike receptor binding domain with a fluorescent protein were created in monomeric and trimeric form as tools for receptor binding studies in cultured cells and animal tissues. Here, site specific N-linked glycosylation in the proteins expressed from GnTI-/- cells is profiled with LC-MS/MS, using electron transfer high-energy collision dissociation
Project description:Glycosylation reactions require activated glycosyl donors in form of nucleotide sugars to drive processes such as post-translational protein modifications, glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi requiring cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters. Here we present the identification of the plant UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (UGNT1) indispensable for the delivery of a substrate for maturation of N-glycans and glycosyl inositol phosphorylceramides (GIPCs). Profiles of N-glycopeptides revealed that UGNT1 loss-of-function mutants are devoid of complex and hybrid N-glycans. Instead, most of the glycol-N-peptide population contained high mannose structures, representing the structure prior to the addition of the first GlcNAc in the Golgi. Our findings emphasize that the reference plant Arabidopsis contains a single UDP-GlcNAc transporter responsible for the maturation of complex N-glycans in the Golgi lumen.
Project description:Coronaviruses (CoVs) are enveloped pathogens causing multiple respiratory disorders in humans with varying severity. Spike protein is one of the major proteins expressed on coronavirus surface, which mediates coronavirus entry into host cells. Spike proteins are extensively glycosylated and the glycans displayed on spike proteins play a key role in host pathogenesis and immune evasion. In this study, we aim to investigate whether glycosylation patterns are conservative at certain glycosites across different coronaviruses and how different host cells impact on the glycosylation profile. We analyzed site-specific glycans of S1 subunit from SARS-CoV and MERS-CoV spike proteins using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. We also compared glycosylation of MERS-CoV spike protein derived from HEK293 and insect cells. Our results show that SARS-CoV S1 and MERS-CoV S1 N-glycosylation presents some common patterns and also reveals the similar O-glycosites locations. Consistent with published data, confirming glycan and glycan subtype in specific positions, our data support that some monoclonal antibodies recognize glycan as part of their target epitope and cross react between SARS Cov and SARS CoV2 spike. The coronavirus spike proteins are highly glycosylated. The glycosylation sites, within each virus, are conserved with few changes over time.
Project description:A workflow for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis has been developed.
Project description:A comprehensive screening of site-specific N-glycopeptides in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation in HCC, has been performed to characterize glycopeptide markers for NASH-related HCCs.