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: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: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: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.
Project description:Nε-lysine acetylation has emerged as a central mechanism to maintain quality control and protein homeostasis within the Endoplasmic Reticulum (ER) and secretory pathway. The ER acetylation machinery includes AT-1/SLC33A1, a membrane transporter that translocates acetyl-CoA from the cytosol to the ER lumen, and ATase1 and ATase2, two ER membrane-bound acetyltransferases that acetylate ER cargo proteins. Dysfunctional AT-1, as caused by loss-of-function mutations or gene duplication events, results in neurodevelopmental or neurodegenerative disorders. Experiments in our lab have demonstrated that these human diseases can be effectively recapitulated in mouse models. In this thesis, we used two models of dysregulated acetyl-CoA flux: AT-1S113R/+, a model of AT-1 haploinsufficiency, and AT-1 sTg, a model of systemic overexpression of AT-1. First, we examined upstream processes of cytosol-to-ER acetyl-CoA flux by evaluating the cytosolic pool of acetyl-CoA. The aberrant AT-1 models demonstrated distinct metabolic reprogramming of lipid metabolism and mitochondria bioenergetics. Dysregulated acetyl-CoA flux resulted in global changes at the level of the proteome and the acetyl-proteome. Second, we examined the downstream consequences of cytosol-to-ER acetyl-CoA flux. Specifically, we investigated the engagement and functional organization of the secretory pathway and used N-glycoproteomics to determine the quality of secreted proteins. Aberrant AT-1 models demonstrated reorganization of the ER, Golgi, and ERGIC, as well as a delay in glycoproteins clearing the Golgi apparatus. Additionally, AT-1 sTg mice showed a marked delay in protein trafficking to the cell surface. The N-glycoproteome revealed significant alterations, highlighting changes in the quality of the secretome.
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:The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To analyze post-translational modifications (PTMs) in the pancreas, native and decellularized tissues from four donors were analyzed.