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: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: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:Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX.This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.
Project description:In this work, we performed a fully descriptive analysis N- and O- linked glycosylation of SARS-COV-2 S glycoprotein. We investigated that dual-functionalized Ti-IMAC material enable the simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293, which will eliminate the signal suppression of neutral glycopeptides to sialyl glycopeptides and improve the glycoform coverage of S protein. We have profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms in dual-functional Ti-IMIAC approach that is 1.6-fold of that in conventional HILIC method. We also identified O-linked glycosylation site that was not found in dual-functional Ti-IMIAC approach. In addition, we have also identified mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of mannose-6-Phosphate on its cell entry.
Project description:HCoV-NL63 is a coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. Of great interest is understanding the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. We utilized an electron-transfer/higher energy collision dissociation ion fragmentation scheme (Frese, C. K. et al., 2013.) in combination with cryo-electron microscopy to resolve the extensive glycan shield that obstructs the protein surface. These glycans provide a structural framework to understanding the accessibility of the protein to antibodies.
Project description:A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography (HILIC). The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly-hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity between 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with “crosstalk” modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase (RP) LC-MS/MS analysis. Between 47-100% of the peptides with more than one phosphate or sialyl- residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.
Project description:Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excretedsecreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses. Here, we employed glycoproteomic and proteomic analyses to determine the glycosylation profile of proteins within the NEJs’ somatic (Som) and ES extracts.