LitCovid-sample-sentences  

PMC:7199903 JSONTXT 39 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T1 0-53 Sentence denotes Site-specific glycan analysis of the SARS-CoV-2 spike
T2 55-91 Sentence denotes SARS-CoV-2 spike protein, elaborated
T3 92-243 Sentence denotes Vaccine development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the trimeric spike protein that initiates infection.
T4 244-307 Sentence denotes Each protomer in the trimeric spike has 22 glycosylation sites.
T5 308-445 Sentence denotes How these sites are glycosylated may affect which cells the virus can infect and could shield some epitopes from antibody neutralization.
T6 446-665 Sentence denotes Watanabe et al. expressed and purified recombinant glycosylated spike trimers, proteolysed them to yield glycopeptides containing a single glycan, and determined the composition of the glycan sites by mass spectrometry.
T7 666-789 Sentence denotes The analysis provides a benchmark that can be used to measure antigen quality as vaccines and antibody tests are developed.
T8 790-811 Sentence denotes Science this issue p.
T9 812-815 Sentence denotes 330
T10 817-936 Sentence denotes A mass spectrometry analysis reveals the glycan composition at all glycosylation sites on the SARS-CoV-2 spike protein.
T11 938-946 Sentence denotes Abstract
T12 947-1163 Sentence denotes The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health.
T13 1164-1325 Sentence denotes Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion.
T14 1326-1460 Sentence denotes The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion.
T15 1461-1590 Sentence denotes Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen.
T16 1591-1685 Sentence denotes This analysis enables mapping of the glycan-processing states across the trimeric viral spike.
T17 1686-1832 Sentence denotes We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.
T18 1834-2015 Sentence denotes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus 2019 (COVID-19) (1, 2), induces fever, severe respiratory illness, and pneumonia.
T19 2016-2197 Sentence denotes SARS-CoV-2 uses an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry (3).
T20 2198-2373 Sentence denotes The S protein is a trimeric class I fusion protein, composed of two functional subunits, responsible for receptor binding (S1 subunit) and membrane fusion (S2 subunit) (4, 5).
T21 2374-2506 Sentence denotes The surface of the envelope spike is dominated by host-derived glycans, with each trimer displaying 66 N-linked glycosylation sites.
T22 2507-2724 Sentence denotes The S protein is a key target in vaccine design efforts (6), and understanding the glycosylation of recombinant viral spikes can reveal fundamental features of viral biology and guide vaccine design strategies (7, 8).
T23 2725-2871 Sentence denotes Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability and shaping viral tropism (9).
T24 2872-3015 Sentence denotes Glycosylation sites are under selective pressure as they facilitate immune evasion by shielding specific epitopes from antibody neutralization.
T25 3016-3161 Sentence denotes However, we note the low mutation rate of SARS-CoV-2 and that as yet, there have been no observed mutations to N-linked glycosylation sites (10).
T26 3162-3260 Sentence denotes Surfaces with an unusually high density of glycans can also enable immune recognition (9, 11, 12).
T27 3261-3386 Sentence denotes The role of glycosylation in camouflaging immunogenic protein epitopes has been studied for other coronaviruses (10, 13, 14).
T28 3387-3506 Sentence denotes Coronaviruses form virions by budding into the lumen of endoplasmic reticulum–Golgi intermediate compartments (15, 16).
T29 3507-3679 Sentence denotes However, observations of complex-type glycans on virally derived material suggests that the viral glycoproteins are subjected to Golgi-resident processing enzymes (13, 17).
T30 3680-3789 Sentence denotes High viral glycan density and local protein architecture can sterically impair the glycan maturation pathway.
T31 3790-4060 Sentence denotes Impaired glycan maturation resulting in the presence of oligomannose-type glycans can be a sensitive reporter of native-like protein architecture (8), and site-specific glycan analysis can be used to compare different immunogens and monitor manufacturing processes (18).
T32 4061-4169 Sentence denotes Additionally, glycosylation can influence the trafficking of recombinant immunogen to germinal centers (19).
T33 4170-4588 Sentence denotes To resolve the site-specific glycosylation of the SARS-CoV-2 S protein and visualize the distribution of glycoforms across the protein surface, we expressed and purified three biological replicates of recombinant soluble material in an identical manner to that which was used to obtain the high-resolution cryo–electron microscopy (cryo-EM) structure, albeit without a glycan-processing blockade using kifunensine (4).
T34 4589-4681 Sentence denotes This variant of the S protein contains all 22 glycans on the SARS-CoV-2 S protein (Fig. 1A).
T35 4682-4938 Sentence denotes Stabilization of the trimeric prefusion structure was achieved by using the 2P stabilizing mutations (20) at residues 986 and 987, a GSAS (Gly-Ser-Ala-Ser) substitution at the furin cleavage site (residues 682 to 685), and a C-terminal trimerization motif.
T36 4939-5011 Sentence denotes This helps to maintain quaternary architecture during glycan processing.
T37 5012-5211 Sentence denotes Before analysis, supernatant containing the recombinant SARS-CoV-2 S was purified by size exclusion chromatography to ensure that only native-like trimeric protein was analyzed (Fig. 1B and fig. S1).
T38 5212-5314 Sentence denotes The trimeric conformation of the purified material was validated by using negative-stain EM (Fig. 1C).
T39 5315-5382 Sentence denotes Fig. 1 Expression and validation of the SARS-CoV-2 S glycoprotein.
T40 5383-5445 Sentence denotes (A) Schematic representation of the SARS-CoV-2 S glycoprotein.
T41 5446-5588 Sentence denotes The positions of N-linked glycosylation sequons (N-X-S/T, where X ≠ P) are shown as branches (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro).
T42 5589-5958 Sentence denotes Protein domains are illustrated: N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). (B) SDS–polyacrylamide gel electrophoresis analysis of the SARS-CoV-2 S protein (indicated by the arrowhead) expressed in human embryonic kidney (HEK) 293F cells.
T43 5959-6202 Sentence denotes Lane 1: filtered supernatant from transfected cells; lane 2: flow-through from StrepTactin resin; lane 3: wash from StrepTactin resin; lane 4: elution from StrepTactin resin. (C) Negative-stain EM 2D class averages of the SARS-CoV-2 S protein.
T44 6203-6389 Sentence denotes 2D class averages of the SARS-CoV-2 S protein are shown, confirming that the protein adopts the trimeric prefusion conformation matching the material used to determine the structure (4).
T45 6390-6543 Sentence denotes To determine the site-specific glycosylation of SARS-CoV-2 S, we used trypsin, chymotrypsin, and α-lytic protease to generate three glycopeptide samples.
T46 6544-6645 Sentence denotes These proteases were selected to generate glycopeptides that contain a single N-linked glycan sequon.
T47 6646-6808 Sentence denotes The glycopeptides were analyzed by liquid chromatography–mass spectrometry, and the glycan compositions were determined for all 22 N-linked glycan sites (Fig. 2).
T48 6809-7021 Sentence denotes To convey the main processing features at each site, the abundances of each glycan are summed into oligomannose-type, hybrid-type, and categories of complex-type glycosylation based on branching and fucosylation.
T49 7022-7139 Sentence denotes The detailed, expanded graphs showing the diverse range of glycan compositions are presented in table S1 and fig. S2.
T50 7140-7218 Sentence denotes Fig. 2 Site-specific N-linked glycosylation of the SARS-CoV-2 S glycoprotein.
T51 7219-7389 Sentence denotes The schematic illustrates the color code for the principal glycan types that can arise along the maturation pathway from oligomannose- to hybrid- to complex-type glycans.
T52 7390-7566 Sentence denotes The graphs summarize quantitative mass spectrometric analysis of the glycan population present at individual N-linked glycosylation sites simplified into categories of glycans.
T53 7567-7883 Sentence denotes The oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) is colored green, afucosylated and fucosylated hybrid-type glycans (hybrid and F hybrid) are dashed pink, and complex glycans are grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) and are colored pink.
T54 7884-7946 Sentence denotes Unoccupancy of an N-linked glycan site is represented in gray.
T55 7947-8008 Sentence denotes The pie charts summarize the quantification of these glycans.
T56 8009-8174 Sentence denotes Glycan sites are colored according to oligomannose-type glycan content, with the glycan sites labeled in green (80 to 100%), orange (30 to 79%), and pink (0 to 29%).
T57 8175-8309 Sentence denotes An extended version of the site-specific analysis showing the heterogeneity within each category can be found in table S1 and fig. S2.
T58 8310-8447 Sentence denotes The bar graphs represent the mean quantities of three biological replicates, with error bars representing the standard error of the mean.
T59 8448-8523 Sentence denotes Two sites on SARS-CoV-2 S are principally oligomannose-type: N234 and N709.
T60 8524-8916 Sentence denotes The predominant oligomannose-type glycan structure observed across the protein, with the exception of N234, is Man5GlcNAc2 (Man, mannose; GlcNAc, N-acetylglucosamine), which demonstrates that these sites are largely accessible to α-1,2-mannosidases but are poor substrates for GlcNAcT-I, which is the gateway enzyme in the formation of hybrid- and complex-type glycans in the Golgi apparatus.
T61 8917-9043 Sentence denotes The stage at which processing is impeded is a signature related to the density and presentation of glycans on the viral spike.
T62 9044-9190 Sentence denotes For example, the more densely glycosylated spikes of HIV-1 Env and Lassa virus (LASV) GPC exhibit numerous sites dominated by Man9GlcNAc2 (21–24).
T63 9191-9313 Sentence denotes A mixture of oligomannose- and complex-type glycans can be found at sites N61, N122, N603, N717, N801, and N1074 (Fig. 2).
T64 9314-9514 Sentence denotes Of the 22 sites on the S protein, 8 contain substantial populations of oligomannose-type glycans, highlighting how the processing of the SARS-CoV-2 S glycans is divergent from host glycoproteins (25).
T65 9515-9587 Sentence denotes The remaining 14 sites are dominated by processed, complex-type glycans.
T66 9588-9762 Sentence denotes Although unoccupied glycosylation sites were detected on SARS-CoV-2 S, when quantified they were revealed to form a very minor component of the total peptide pool (table S2).
T67 9763-9928 Sentence denotes In HIV-1 immunogen research, the holes generated by unoccupied glycan sites have been shown to be immunogenic and potentially give rise to distracting epitopes (26).
T68 9929-10093 Sentence denotes The high occupancy of N-linked glycan sequons of SARS-CoV-2 S indicates that recombinant immunogens will not require further optimization to enhance site occupancy.
T69 10094-10343 Sentence denotes Using the cryo-EM structure of the trimeric SARS-CoV-2 S protein [Protein Data Bank (PDB) ID 6VSB] (4), we mapped the glycosylation status of the coronavirus spike mimetic onto the experimentally determined three-dimensional (3D) structure (Fig. 3).
T70 10344-10499 Sentence denotes This combined mass spectrometric and cryo-EM analysis reveals how the N-linked glycans occlude distinct regions across the surface of the SARS-CoV-2 spike.
T71 10500-10565 Sentence denotes Fig. 3 Structure-based mapping of SARS-CoV-2 S N-linked glycans.
T72 10566-10779 Sentence denotes Representative glycans are modeled onto the prefusion structure of the trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), with one RBD in the “up” conformation and the other two RBDs in the “down” conformation.
T73 10780-10860 Sentence denotes The glycans are colored according to oligomannose content as defined by the key.
T74 10861-10919 Sentence denotes ACE2 receptor binding sites are highlighted in light blue.
T75 10920-11039 Sentence denotes The S1 and S2 subunits are rendered with translucent surface representation, colored light and dark gray, respectively.
T76 11040-11210 Sentence denotes The flexible loops on which the N74 and N149 glycan sites reside are represented as gray dashed lines, with glycan sites on the loops mapped at their approximate regions.
T77 11211-11417 Sentence denotes Shielding of the receptor binding sites on the SARS-CoV-2 spike by proximal glycosylation sites (N165, N234, N343) can be observed, especially when the receptor binding domain is in the “down” conformation.
T78 11418-11619 Sentence denotes The shielding of receptor binding sites by glycans is a common feature of viral glycoproteins, as observed on SARS-CoV-1 S (10, 13), HIV-1 Env (27), influenza hemagglutinin (28, 29), and LASV GPC (24).
T79 11620-11910 Sentence denotes Given the functional constraints of receptor binding sites and the resulting low mutation rates of these residues, there is likely selective pressure to use N-linked glycans to camouflage one of the most conserved and potentially vulnerable areas of their respective glycoproteins (30, 31).
T80 11911-11998 Sentence denotes We note the dispersion of oligomannose-type glycans across both the S1 and S2 subunits.
T81 11999-12196 Sentence denotes This is in contrast to other viral glycoproteins; for example, the dense glycan clusters in several strains of HIV-1 Env induce oligomannose-type glycans that are recognized by antibodies (32, 33).
T82 12197-12420 Sentence denotes In SARS-CoV-2 S, the oligomannose-type structures are likely protected by the protein component, as exemplified by the N234 glycan, which is partially sandwiched between the N-terminal and receptor binding domains (Fig. 3).
T83 12421-12620 Sentence denotes We characterized the N-linked glycans on extended flexible loop structures (N74 and N149) and at the membrane-proximal C terminus (N1158, N1173, N1194) that were not resolved in the cryo-EM maps (4).
T84 12621-12726 Sentence denotes These were determined to be complex-type glycans, consistent with steric accessibility of these residues.
T85 12727-12886 Sentence denotes Whereas the oligomannose-type glycan content (28%) (table S2) is above that observed on typical host glycoproteins, it is lower than other viral glycoproteins.
T86 12887-13027 Sentence denotes For example, one of the most densely glycosylated viral spike proteins is HIV-1 Env, which exhibits ~60% oligomannose-type glycans (21, 34).
T87 13028-13290 Sentence denotes This suggests that the SARS-CoV-2 S protein is less densely glycosylated and that the glycans form less of a shield compared with other viral glycoproteins, including HIV-1 Env and LASV GPC, which may be beneficial for the elicitation of neutralizing antibodies.
T88 13291-13530 Sentence denotes Additionally, the processing of complex-type glycans is an important consideration in immunogen engineering, especially considering that epitopes of neutralizing antibodies against SARS-CoV-2 S can contain fucosylated glycans at N343 (35).
T89 13531-13682 Sentence denotes Across the 22 N-linked glycosylation sites, 52% are fucosylated and 15% of the glycans contain at least one sialic acid residue (table S2 and fig. S3).
T90 13683-13789 Sentence denotes Our analysis reveals that N343 is highly fucosylated with 98% of detected glycans bearing fucose residues.
T91 13790-13876 Sentence denotes Glycan modifications can be heavily influenced by the cellular expression system used.
T92 13877-14194 Sentence denotes We have previously demonstrated for HIV-1 Env glycosylation that the processing of complex-type glycans is driven by the producer cell but that the levels of oligomannose-type glycans were largely independent of the expression system and are much more closely related to the protein structure and glycan density (36).
T93 14195-14350 Sentence denotes Highly dense glycan shields, such as those observed on LASV GPC and HIV-1 Env, feature so-called mannose clusters (22, 24) on the protein surface (Fig. 4).
T94 14351-14565 Sentence denotes Whereas small mannose-type clusters have been characterized on the S1 subunit of Middle East respiratory syndrome (MERS)–CoV S (10), no such phenomenon has been observed for the SARS-CoV-1 or SARS-CoV-2 S proteins.
T95 14566-14790 Sentence denotes The site-specific glycosylation analysis reported here suggests that the glycan shield of SARS-CoV-2 S is consistent with other coronaviruses and similarly exhibits numerous vulnerabilities throughout the glycan shield (10).
T96 14791-15028 Sentence denotes Last, we detected trace levels of O-linked glycosylation at Thr323/Ser325 (T323/S325), with over 99% of these sites unmodified (fig. S4), suggesting that O-linked glycosylation of this region is minimal when the structure is native-like.
T97 15029-15077 Sentence denotes Fig. 4 Underprocessing of viral glycan shields.
T98 15078-15185 Sentence denotes From left to right, MERS-CoV S (10), SARS-CoV-1 S (10), SARS-CoV-2 S, LASV GPC (24), and HIV-1 Env (8, 21).
T99 15186-15278 Sentence denotes Site-specific N-linked glycan oligomannose quantifications are colored according to the key.
T100 15279-15454 Sentence denotes All glycoproteins were expressed as soluble trimers in HEK 293F cells apart from LASV GPC, which was derived from virus-like particles from Madin-Darby canine kidney II cells.
T101 15455-15610 Sentence denotes Our glycosylation analysis of SARS-CoV-2 offers a detailed benchmark of site-specific glycan signatures characteristic of a natively folded trimeric spike.
T102 15611-15870 Sentence denotes As an increasing number of glycoprotein-based vaccine candidates are being developed, their detailed glycan analysis offers a route for comparing immunogen integrity and will also be important to monitor as manufacturing processes are scaled for clinical use.
T103 15871-15998 Sentence denotes Glycan profiling will therefore also be an important measure of antigen quality in the manufacture of serological testing kits.
T104 15999-16161 Sentence denotes Last, with the advent of nucleotide-based vaccines, it will be important to understand how those delivery mechanisms affect immunogen processing and presentation.
T105 16163-16178 Sentence denotes Acknowledgments
T106 16179-16190 Sentence denotes We thank M.
T107 16191-16203 Sentence denotes Dixon and M.
T108 16204-16314 Sentence denotes Gowland-Pryde for supporting our work on this project during the difficulties arising from the pandemic and G.
T109 16315-16359 Sentence denotes Ould for critical reading of the manuscript.
T110 16360-16368 Sentence denotes Funding:
T111 16369-16675 Sentence denotes This work was funded by the International AIDS Vaccine Initiative, Bill and Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery (OPP1084519 and 1196345 to M.C.), the NIAID (R01-AI127521 to J.S.M.), and the Scripps Consortium for HIV Vaccine Development (CHAVD) (AI144462 to M.C.).
T112 16676-16779 Sentence denotes M.C. is a Supernumerary Fellow of Oriel College, Oxford, and professor adjunct at Scripps Research, CA.
T113 16780-16876 Sentence denotes Author contributions: Y.W. and J.D.A. performed mass spectrometry experiments and analyzed data.
T114 16877-16908 Sentence denotes Y.W. built glycosylated models.
T115 16909-16948 Sentence denotes J.S.M. and M.C. supervised experiments.
T116 16949-17004 Sentence denotes Y.W., J.D.A., and D.W. expressed and purified proteins.
T117 17005-17077 Sentence denotes Y.W., J.D.A., and M.C. wrote the manuscript with input from all authors.
T118 17078-17151 Sentence denotes Competing interests: J.S.M. is an inventor on U.S. patent application no.
T119 17152-17283 Sentence denotes 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”), and D.W. and J.S.M. are inventors on U.S. patent application no.
T120 17284-17317 Sentence denotes 62/972,886 (“2019-nCoV Vaccine”).
T121 17318-17350 Sentence denotes Data and materials availability:
T122 17351-17439 Sentence denotes Mass spectrometry raw files have been deposited in the MassIVE proteomics database (37).
T123 17440-17552 Sentence denotes The plasmid is available from J.S.M. under a material transfer agreement with The University of Texas at Austin.
T124 17553-17774 Sentence denotes This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
T125 17775-17858 Sentence denotes To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.
T126 17859-18063 Sentence denotes This license does not apply to figures, photos, artwork, or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.
T127 18065-18088 Sentence denotes Supplementary Materials
T128 18089-18142 Sentence denotes science.sciencemag.org/content/369/6501/330/suppl/DC1
T129 18143-18164 Sentence denotes Materials and Methods
T130 18165-18170 Sentence denotes Figs.
T131 18171-18179 Sentence denotes S1 to S4
T132 18180-18196 Sentence denotes Tables S1 and S2
T133 18197-18216 Sentence denotes References (38, 39)
T134 18217-18247 Sentence denotes MDAR Reproducibility Checklist
T135 18248-18255 Sentence denotes Data S1
T136 18257-18314 Sentence denotes View/request a protocol for this paper from Bio-protocol.