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1.
Proc Natl Acad Sci U S A ; 119(28): e2119761119, 2022 Jul 12.
Article in English | MEDLINE | ID: covidwho-1900767

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein is the prime target for vaccines, diagnostics, and therapeutic antibodies against the virus. While anchored in the viral envelope, for effective virulence, the spike needs to maintain structural flexibility to recognize the host cell surface receptors and bind to them, a property that can heavily depend upon the dynamics of the unresolved domains, most prominently the stalk. Construction of the complete, membrane-bound spike model and the description of its dynamics are critical steps in understanding the inner working of this key element of the viral infection by SARS-CoV-2. Combining homology modeling, protein-protein docking, and molecular dynamics (MD) simulations, we have developed a full spike structure in a native membrane. Multimicrosecond MD simulations of this model, the longest known single trajectory of the full spike, reveal conformational dynamics employed by the protein to explore the surface of the host cell. In agreement with cryogenic electron microscopy (cryo-EM), three flexible hinges in the stalk allow for global conformational heterogeneity of spike in the fully glycosylated system mediated by glycan-glycan and glycan-lipid interactions. The dynamical range of the spike is considerably reduced in its nonglycosylated form, confining the area explored by the spike on the host cell surface. Furthermore, palmitoylation of the membrane domain amplifies the local curvature that may prime the fusion. We show that the identified hinge regions are highly conserved in SARS coronaviruses, highlighting their functional importance in enhancing viral infection, and thereby, provide points for discovery of alternative therapeutics against the virus.


Subject(s)
COVID-19 , Host Microbial Interactions , Protein Processing, Post-Translational , Receptors, Cell Surface , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , COVID-19/virology , Glycosylation , Humans , Polysaccharides , Protein Binding , Receptors, Cell Surface/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
Proteomics ; 22(15-16): e2100322, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1885450

ABSTRACT

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.


Subject(s)
COVID-19 , SARS-CoV-2 , Glycosylation , Humans , Mass Spectrometry/methods , Polysaccharides/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
3.
Anal Chem ; 94(15): 5776-5784, 2022 04 19.
Article in English | MEDLINE | ID: covidwho-1882709

ABSTRACT

Characterization of protein glycosylation by tandem mass spectrometry remains challenging owing to the vast diversity of oligosaccharides bound to proteins, the variation in monosaccharide linkage patterns, and the lability of the linkage between the glycan and protein. Here, we have adapted an HCD-triggered-ultraviolet photodissociation (UVPD) approach for the simultaneous localization of glycosites and full characterization of both glycan compositions and intersaccharide linkages, the latter provided by extensive cross-ring cleavages enabled by UVPD. The method is applied to study glycan compositions based on analysis of glycopeptides from proteolytic digestion of recombinant human coronaviruse spike proteins from SARS-CoV-2 and HKU1. UVPD reveals unique intersaccharide linkage information and is leveraged to localize N-linked glycoforms with confidence.


Subject(s)
COVID-19 , Viral Proteins , Glycosylation , Humans , Polysaccharides/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Tandem Mass Spectrometry/methods , Ultraviolet Rays
5.
Curr Opin Struct Biol ; 73: 102348, 2022 04.
Article in English | MEDLINE | ID: covidwho-1852025

ABSTRACT

Vaccine-induced immunity is expected to target the native antigens expressed by the pathogens. Therefore, it is highly important to generate vaccine antigens that are immunologically indistinguishable from the native antigens. Nucleic acid vaccines, comprised of DNA, mRNA, or recombinant viral vector vaccines, introduce the genetic material encoding the antigenic protein for the host to express. Because these proteins will undergo host posttranslational modifications, host glycosylation can potentially alter the structure and immunological efficacy of the antigen. In this review, we discuss the potential impact of host protein glycosylation on the immune responses generated by nucleic acid vaccines against bacterial and viral pathogens.


Subject(s)
Viral Vaccines , Antigens , Glycosylation , Protein Processing, Post-Translational , Viral Vaccines/genetics
6.
J Med Virol ; 94(9): 4181-4192, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1844141

ABSTRACT

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites.


Subject(s)
SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Glycosylation , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
7.
J Am Chem Soc ; 144(20): 9057-9065, 2022 05 25.
Article in English | MEDLINE | ID: covidwho-1839492

ABSTRACT

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.


Subject(s)
COVID-19 , Glycopeptides , SARS-CoV-2 , Glycopeptides/chemical synthesis , Glycopeptides/chemistry , Glycoproteins/chemistry , Glycosylation , Humans , Peptides/metabolism , SARS-CoV-2/chemistry
8.
EBioMedicine ; 78: 103957, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1828375

ABSTRACT

BACKGROUND: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific IgG1 has been described as a hallmark of severe coronavirus disease 2019 (COVID-19) and may lead to activation of macrophages via immune complexes thereby promoting inflammatory responses, altogether suggesting involvement of IgG1 Fc glycosylation modulated immune mechanisms in COVID-19. METHODS: In this prospective, observational single center cohort study, IgG1 Fc glycosylation was analyzed by liquid chromatography-mass spectrometry following affinity capturing from serial plasma samples of 159 SARS-CoV-2 infected hospitalized patients. FINDINGS: At baseline close to disease onset, anti-S IgG1 glycosylation was highly skewed when compared to total plasma IgG1. A rapid, general reduction in glycosylation skewing was observed during the disease course. Low anti-S IgG1 galactosylation and sialylation as well as high bisection were early hallmarks of disease severity, whilst high galactosylation and sialylation and low bisection were found in patients with low disease severity. In line with these observations, anti-S IgG1 glycosylation correlated with various inflammatory markers. INTERPRETATION: Association of low galactosylation, sialylation as well as high bisection with disease severity and inflammatory markers suggests that further studies are needed to understand how anti-S IgG1 glycosylation may contribute to disease mechanism and to evaluate its biomarker potential. FUNDING: This project received funding from the European Commission's Horizon2020 research and innovation program for H2020-MSCA-ITN IMforFUTURE, under grant agreement number 721815, and supported by Crowdfunding Wake Up To Corona, organized by the Leiden University Fund.


Subject(s)
COVID-19 , Biomarkers , Cohort Studies , Glycosylation , Humans , Immunoglobulin Fc Fragments , Immunoglobulin G , Prospective Studies , SARS-CoV-2
9.
Comput Biol Chem ; 98: 107668, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1828156

ABSTRACT

The emergence of COVID-19 caused by SARS-CoV-2 and its spread since 2019 represents the major public health problem worldwide nowadays, which has generated a high number of infections and deaths. The spike protein (S protein) is the most studied protein of SARS-CoV-2, and key to host-cell entry through ACE2 receptor. This protein presents a large pattern of glycosylations with important roles in immunity and infection mechanisms. Therefore, understanding key aspects of the molecular mechanisms of these structures, during drug recognition in SARS-CoV-2, may contribute to therapeutic alternatives. In this work, we explored the impact of glycosylations on the drug recognition on two domains of the S protein, the receptor-binding domain (RBD) and the N-terminal domain (NTD) through molecular dynamics simulations and computational biophysics analysis. Our results show that glycosylations in the S protein induce structural stability and changes in rigidity/flexibility related to the number of glycosylations in the structure. These structural changes are important for its biological activity as well as the correct interaction of ligands in the RBD and NTD regions. Additionally, we evidenced a roto-translation phenomenon in the interaction of the ligand with RBD in the absence of glycosylation, which disappears due to the influence of glycosylation and the convergence of metastable states in RBM. Similarly, glycosylations in NTD promote an induced fit phenomenon, which is not observed in the absence of glycosylations; this process is decisive for the activity of the ligand at the cryptic site. Altogether, these results provide an explanation of glycosylation relevance in biophysical properties and drug recognition to S protein of SARS-CoV-2, which must be considered in the rational drug development and virtual screening targeting S protein.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Glycoproteins , Glycosylation , Humans , Ligands , Molecular Dynamics Simulation , Protein Binding , Spike Glycoprotein, Coronavirus/metabolism
10.
Virulence ; 13(1): 670-683, 2022 12.
Article in English | MEDLINE | ID: covidwho-1791073

ABSTRACT

Glycans are among the most important cell molecular components. However, given their structural diversity, their functions have not been fully explored. Glycosylation is a vital post-translational modification for various proteins. Many bacteria and viruses rely on N-linked and O-linked glycosylation to perform critical biological functions. The diverse functions of glycosylation on viral proteins during viral infections, including Dengue, Zika, influenza, and human immunodeficiency viruses as well as coronaviruses have been reported. N-linked glycosylation is the most common form of protein modification, and it modulates folding, transportation and receptor binding. Compared to N-linked glycosylation, the functions of O-linked viral protein glycosylation have not been comprehensively evaluated. In this review, we summarize findings on viral protein glycosylation, with particular attention to studies on N-linked glycosylation in viral life cycles. This review informs the development of virus-specific vaccines or inhibitors.


Subject(s)
Zika Virus Infection , Zika Virus , Glycosylation , Host Microbial Interactions , Humans , Protein Processing, Post-Translational , Viral Proteins/metabolism , Virulence , Zika Virus/metabolism
12.
Nat Methods ; 19(4): 384-387, 2022 04.
Article in English | MEDLINE | ID: covidwho-1788308
13.
Eur J Immunol ; 52(6): 946-957, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1750362

ABSTRACT

The nature of the immune responses associated with COVID-19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID-19. We have analyzed IgG Fc N-glycan traits of 82 SARS-CoV-2+ unvaccinated patients, at diagnosis, by nano-LC-ESI-MS. We determined the impact of IgG Fc glyco-variations in the induction of NK cells activation, further evaluating the association between IgG Fc N-glycans and disease severity/prognosis. We found that SARS-CoV-2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID-19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID-19 patients appears to induce NK cells activation associated with increased release of IFN-γ and TNF-α, which indicates the presence of pro-inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.


Subject(s)
COVID-19 , Biomarkers , COVID-19/diagnosis , COVID-19 Testing , Galactose , Glycosylation , Humans , Immunoglobulin Fc Fragments , Immunoglobulin G , Polysaccharides , SARS-CoV-2 , Severity of Illness Index
14.
Sci Rep ; 12(1): 4269, 2022 03 11.
Article in English | MEDLINE | ID: covidwho-1740479

ABSTRACT

It has been reported that patients diagnosed with COVID-19 become critically ill primarily around the time of activation of the adaptive immune response. However the role of antibodies in the worsening of disease is not obvious. Higher titers of anti-spike immunoglobulin IgG1 associated with low fucosylation of the antibody Fc tail have been associated to excessive inflammatory response. In contrast it has been also reported that NP-, S-, RBD- specific IgA, IgG, and IgM are not associated with SARS-CoV-2 viral load, indicating that there is no obvious correlation between antibody response and viral antigen detection. In the present work the micro-Fourier-transform infrared reflectance spectroscopy (micro-FTIR) was employed to investigate blood serum samples of healthy and COVID-19-ill (mild or oligosymptomatic) individuals (82 healthcare workers volunteers in "Instituto de Infectologia Emilio Ribas", São Paulo, Brazil). The molecular-level-sensitive, multiplexing quantitative and qualitative FTIR data probed on 1 µL of dried biofluid was compared to signal-to-cutoff index of chemiluminescent immunoassays CLIA and ELISA (IgG antibodies against SARS-CoV-2). Our main result indicated that 1702-1785 [Formula: see text] spectral window (carbonyl C=O vibration) is a spectral marker of the degree of IgG glycosylation, allowing to probe distinctive sub-populations of COVID-19 patients, depending on their degree of severity. The specificity was 87.5 % while the detection rate of true positive was 100%. The computed area under the receiver operating curve was equivalent to CLIA, ELISA and other ATR-FTIR methods ([Formula: see text]). In summary, overall discrimination of healthy and COVID-19 individuals and severity prediction as well could be potentially implemented using micro-FTIR reflectance spectroscopy on blood serum samples. Considering the minimal and reagent-free sample preparation procedures combined to fast (few minutes) outcome of FTIR we can state that this technology is suitable for fast screening of immune response of individuals with COVID-19. It would be an important tool in prospective studies, helping investigate the physiology of the asymptomatic, oligosymptomatic, or severe individuals and measure the extension of infection dissemination in patients.


Subject(s)
COVID-19/metabolism , Immunoglobulin G/metabolism , SARS-CoV-2/immunology , Spectroscopy, Fourier Transform Infrared/methods , Adult , Antibodies, Viral/immunology , COVID-19/diagnostic imaging , COVID-19/immunology , COVID-19 Testing/methods , Enzyme-Linked Immunosorbent Assay , Female , Glycosylation , Humans , Luminescent Measurements , Male , Middle Aged , Patient Acuity , Reproducibility of Results , Sensitivity and Specificity , Spectroscopy, Fourier Transform Infrared/instrumentation , Viral Load
15.
Int J Mol Sci ; 23(5)2022 Feb 25.
Article in English | MEDLINE | ID: covidwho-1736943

ABSTRACT

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.


Subject(s)
COVID-19/metabolism , Erythrocytes/metabolism , SARS-CoV-2/metabolism , Sialoglycoproteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Basigin/metabolism , Binding Sites , COVID-19/virology , Glycosylation , Hemagglutination , Hemagglutinins, Viral/metabolism , Humans , N-Acetylneuraminic Acid/metabolism , Polysaccharides/metabolism , Protein Binding , SARS-CoV-2/physiology , Viral Fusion Proteins/metabolism , Virus Internalization
16.
Int J Mol Sci ; 23(5)2022 Feb 23.
Article in English | MEDLINE | ID: covidwho-1715405

ABSTRACT

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glutathione/analogs & derivatives , Hesperidin/therapeutic use , Lactoylglutathione Lyase/metabolism , Neoplasms, Experimental/drug therapy , Resveratrol/therapeutic use , Animals , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/physiopathology , Drug Therapy, Combination , Enzyme Induction/drug effects , Glutathione/chemistry , Glutathione/therapeutic use , Glycosylation/drug effects , Hesperidin/chemistry , Humans , Insulin Resistance/physiology , Lactoylglutathione Lyase/antagonists & inhibitors , Mice , Molecular Structure , Neoplasms, Experimental/metabolism , Obesity/drug therapy , Obesity/metabolism , Obesity/physiopathology , Pyruvaldehyde/chemistry , Pyruvaldehyde/metabolism , Resveratrol/chemistry
17.
Int J Mol Sci ; 23(5)2022 Feb 23.
Article in English | MEDLINE | ID: covidwho-1715404

ABSTRACT

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.


Subject(s)
Apolipoproteins/metabolism , Caspases/metabolism , Complement System Proteins/metabolism , Cytokines/metabolism , alpha 1-Antitrypsin/metabolism , Binding Sites/genetics , COVID-19/metabolism , COVID-19/virology , Glycosylation , Humans , Mutation , Protein Binding , Protein Domains , SARS-CoV-2/physiology , alpha 1-Antitrypsin/chemistry , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin Deficiency/metabolism
18.
Pharmacol Res Perspect ; 10(2): e00940, 2022 04.
Article in English | MEDLINE | ID: covidwho-1712175

ABSTRACT

Anti-proinflammatory cytokine therapies against interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2-deoxy-d-glucose (2-DG), a simple monosaccharide, attenuated cellular responses to IL-6 by inhibiting N-linked glycosylation of the IL-6 receptor gp130. Aglycoforms of gp130 did not bind to IL-6 or activate downstream intracellular signals that included Janus kinases. 2-DG completely inhibited dextran sodium sulfate-induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin-induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL-6. We also found that 2-DG inhibited signals for other proinflammatory cytokines such as TNF-α, IL-1ß, and interferon -γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2-DG prevented LPS shock, a model for a cytokine storm, and LPS-induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID-19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Deoxyglucose/pharmacology , Glycosylation/drug effects , Inflammation/prevention & control , Receptors, Cytokine/drug effects , Animals , Cells, Cultured , Cytokine Receptor gp130/antagonists & inhibitors , Cytokine Receptor gp130/metabolism , Cytokine Release Syndrome/prevention & control , Cytokines/metabolism , Inflammation/chemically induced , Janus Kinases/drug effects , Lipopolysaccharides , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Cytokine/immunology , Receptors, Cytokine/metabolism , Receptors, Interleukin-6/antagonists & inhibitors , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/metabolism
19.
Life Sci ; 295: 120411, 2022 Apr 15.
Article in English | MEDLINE | ID: covidwho-1683412

ABSTRACT

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.


Subject(s)
COVID-19/drug therapy , Deoxyglucose/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Adenosine Triphosphate/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/metabolism , COVID-19/virology , Cell Proliferation/drug effects , Cell Survival/drug effects , Chlorocebus aethiops , Glucose/metabolism , Glycolysis/drug effects , Glycosylation , Host-Pathogen Interactions/drug effects , Mannose/pharmacology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virion/drug effects , Virion/pathogenicity , Virus Replication/drug effects
20.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: covidwho-1684242

ABSTRACT

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.


Subject(s)
COVID-19 Vaccines/immunology , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Viral/immunology , Antibody Formation , CD8-Positive T-Lymphocytes/immunology , Cytokines/metabolism , Glycosylation , HEK293 Cells , Histocompatibility Antigens/metabolism , Humans , Immunity , Mice, Inbred BALB C , Unfolded Protein Response , Vaccines, Synthetic/immunology , /immunology
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