Pathogen-sugar interactions revealed by universal saturation transfer analysis
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Abstract
Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an “end-on” manner. uSTA-guided modeling and a high-resolution cryo–electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.
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SciScore for 10.1101/2021.04.14.439284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). anti-Human IgG-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expi293 cells (Thermofisher Scientific) were used to express the Spike-Bap protein. Expi293suggested: RRID:CVCL_D615)Pseudoviral Cell-entry Asssay: A spike-expressing lentivirus in HEK … SciScore for 10.1101/2021.04.14.439284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). anti-Human IgG-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expi293 cells (Thermofisher Scientific) were used to express the Spike-Bap protein. Expi293suggested: RRID:CVCL_D615)Pseudoviral Cell-entry Asssay: A spike-expressing lentivirus in HEK 293T cells was generated using a two-plasmid system. HEK 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). MDCKsuggested: NoneSoftware and Algorithms Sentences Resources The values we obtain performing this analysis on BSA/Trp closely match those measured by ITC, and the values we measure for ligand 2 and SPIKE are in good agreement with those measured by SPR as described in the text. SPIKEsuggested: (SPIKE, RRID:SCR_010466)The uSTA analysis pipeline then provides a user with a report that shows the results of the various stages of analysis, and uses pymol to render the surfaces. pymolsuggested: (PyMOL, RRID:SCR_000305)Genetic analysis of clinical samples: Variant calling: Reads were mapped to the hg19 reference genome by the Burrow-Wheeler aligner BWA. BWAsuggested: (BWA, RRID:SCR_010910)Variants were annotated by ANNOVAR. ANNOVARsuggested: (ANNOVAR, RRID:SCR_012821)All data pre-processing and the RFE procedure was coded in Python; the LR model was used, as included, in the scikit-learn module with the liblinear coordinate descent optimization algorithm. Pythonsuggested: (IPython, RRID:SCR_001658)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04549831 Recruiting Genetic Bases of COVID-19 Clinical Variability Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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