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LitCovid-PubTator

LitCovid-PD-FMA-UBERON

LitCovid-PD-UBERON

{"project":"LitCovid-PD-UBERON","denotations":[{"id":"T3","span":{"begin":842,"end":846},"obj":"Body_part"}],"attributes":[{"id":"A3","pred":"uberon_id","subj":"T3","obj":"http://purl.obolibrary.org/obo/UBERON_0000033"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-sample-MedDRA

{"project":"LitCovid-sample-MedDRA","denotations":[{"id":"T3","span":{"begin":3580,"end":3594},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"}],"attributes":[{"id":"A3","pred":"meddra_id","subj":"T3","obj":"http://purl.bioontology.org/ontology/MEDDRA/10069604"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sample-PD-IDO

{"project":"LitCovid-sample-PD-IDO","denotations":[{"id":"T26","span":{"begin":183,"end":188},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T27","span":{"begin":1176,"end":1181},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T28","span":{"begin":1222,"end":1226},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T29","span":{"begin":1577,"end":1581},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T30","span":{"begin":1734,"end":1738},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T31","span":{"begin":3291,"end":3296},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T32","span":{"begin":3323,"end":3328},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T33","span":{"begin":4798,"end":4806},"obj":"http://purl.obolibrary.org/obo/IDO_0000528"},{"id":"T34","span":{"begin":4973,"end":4977},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T35","span":{"begin":5066,"end":5071},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"},{"id":"T36","span":{"begin":5230,"end":5243},"obj":"http://purl.obolibrary.org/obo/BFO_0000034"},{"id":"T37","span":{"begin":5371,"end":5378},"obj":"http://purl.obolibrary.org/obo/NCBITaxon_10239"},{"id":"T38","span":{"begin":5468,"end":5481},"obj":"http://purl.obolibrary.org/obo/BFO_0000034"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sample-Enju

LitCovid-sample-PD-FMA

LitCovid-sample-CHEBI

LitCovid-sample-PD-NCBITaxon

LitCovid-sample-sentences

LitCovid-sample-PD-UBERON

{"project":"LitCovid-sample-PD-UBERON","denotations":[{"id":"T3","span":{"begin":842,"end":846},"obj":"Body_part"}],"attributes":[{"id":"A3","pred":"uberon_id","subj":"T3","obj":"http://purl.obolibrary.org/obo/UBERON_0000033"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sample-PD-MONDO

LitCovid-sample-Pubtator

LitCovid-sample-UniProt

LitCovid-sample-PD-MAT

{"project":"LitCovid-sample-PD-MAT","denotations":[{"id":"T1","span":{"begin":842,"end":846},"obj":"http://purl.obolibrary.org/obo/MAT_0000294"},{"id":"T2","span":{"begin":2371,"end":2379},"obj":"http://purl.obolibrary.org/obo/MAT_0000086"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sample-PD-GO-BP-0

{"project":"LitCovid-sample-PD-GO-BP-0","denotations":[{"id":"T26","span":{"begin":169,"end":182},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T27","span":{"begin":1162,"end":1175},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T28","span":{"begin":2401,"end":2413},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T29","span":{"begin":2613,"end":2625},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T30","span":{"begin":2630,"end":2641},"obj":"http://purl.obolibrary.org/obo/GO_0097503"},{"id":"T31","span":{"begin":2794,"end":2807},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T32","span":{"begin":3054,"end":3067},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T33","span":{"begin":4692,"end":4705},"obj":"http://purl.obolibrary.org/obo/GO_0045087"},{"id":"T34","span":{"begin":5407,"end":5439},"obj":"http://purl.obolibrary.org/obo/GO_0043687"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sample-GO-BP

{"project":"LitCovid-sample-GO-BP","denotations":[{"id":"T23","span":{"begin":169,"end":182},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T24","span":{"begin":1162,"end":1175},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T25","span":{"begin":2401,"end":2413},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T26","span":{"begin":2613,"end":2625},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T27","span":{"begin":2630,"end":2641},"obj":"http://purl.obolibrary.org/obo/GO_0097503"},{"id":"T28","span":{"begin":2794,"end":2807},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T29","span":{"begin":3054,"end":3067},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T30","span":{"begin":4692,"end":4705},"obj":"http://purl.obolibrary.org/obo/GO_0045087"},{"id":"T31","span":{"begin":5407,"end":5439},"obj":"http://purl.obolibrary.org/obo/GO_0043687"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-PD-GO-BP

{"project":"LitCovid-PD-GO-BP","denotations":[{"id":"T26","span":{"begin":160,"end":182},"obj":"http://purl.obolibrary.org/obo/GO_0006487"},{"id":"T27","span":{"begin":169,"end":182},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T28","span":{"begin":1162,"end":1175},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T29","span":{"begin":2401,"end":2413},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T30","span":{"begin":2613,"end":2625},"obj":"http://purl.obolibrary.org/obo/GO_0036065"},{"id":"T31","span":{"begin":2630,"end":2641},"obj":"http://purl.obolibrary.org/obo/GO_0097503"},{"id":"T32","span":{"begin":2794,"end":2807},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T33","span":{"begin":3054,"end":3067},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T34","span":{"begin":4692,"end":4705},"obj":"http://purl.obolibrary.org/obo/GO_0045087"},{"id":"T35","span":{"begin":5407,"end":5439},"obj":"http://purl.obolibrary.org/obo/GO_0043687"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

LitCovid-sentences

LitCovid-sample-Glycan

{"project":"LitCovid-sample-Glycan","denotations":[{"id":"T46","span":{"begin":1302,"end":1309},"obj":"http://rdf.glyconavi.org/CarTNa/CarTNa218/trivialname"},{"id":"T47","span":{"begin":4332,"end":4339},"obj":"http://rdf.glyconavi.org/CarTNa/CarTNa218/trivialname"},{"id":"T48","span":{"begin":4380,"end":4387},"obj":"http://rdf.glyconavi.org/CarTNa/CarTNa218/trivialname"}],"text":"Clustering of underprocessed glycans on MERS S\nWe subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS). This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b). This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a). We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28. As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4). The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.\nFig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.\nPurified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins. Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS. Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans. The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid \u0026 F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink). Left to right; least processed to most processed. The pie charts summarise the quantification of these glycans. Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.\nFig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.\nThe modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins. The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins. The glycans are colored according to oligomannose content, as defined by the key. DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue. The S1 and S2 subunits are colored light grey and dark grey, respectively.\nFig. 4 Amino-acid sequence diversification across SARS and MERS spikes.\na Amino-acid diversity in SARS and MERS S gene sequences. Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively). The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11. S proteins are presented as backbone traces with residues colored according to amino-acid diversity. Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.\nInterestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed. We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS. Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.\nGiven that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50. We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality."}

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