PMC:7443692 / 9613-13789 JSON TXT

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LitCovid-sample-MedDRA

{"project":"LitCovid-sample-MedDRA","denotations":[{"id":"T10","span":{"begin":2953,"end":2963},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"},{"id":"T11","span":{"begin":3144,"end":3150},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"}],"attributes":[{"id":"A10","pred":"meddra_id","subj":"T10","obj":"http://purl.bioontology.org/ontology/MEDDRA/10069374"},{"id":"A11","pred":"meddra_id","subj":"T11","obj":"http://purl.bioontology.org/ontology/MEDDRA/10047890"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-CHEBI

LitCovid-sample-PD-NCBITaxon

LitCovid-sample-sentences

{"project":"LitCovid-sample-sentences","denotations":[{"id":"T49","span":{"begin":0,"end":109},"obj":"Sentence"},{"id":"T50","span":{"begin":110,"end":616},"obj":"Sentence"},{"id":"T51","span":{"begin":617,"end":745},"obj":"Sentence"},{"id":"T52","span":{"begin":746,"end":945},"obj":"Sentence"},{"id":"T53","span":{"begin":946,"end":1174},"obj":"Sentence"},{"id":"T54","span":{"begin":1175,"end":1422},"obj":"Sentence"},{"id":"T55","span":{"begin":1423,"end":1756},"obj":"Sentence"},{"id":"T56","span":{"begin":1757,"end":1976},"obj":"Sentence"},{"id":"T57","span":{"begin":1977,"end":2218},"obj":"Sentence"},{"id":"T58","span":{"begin":2219,"end":2340},"obj":"Sentence"},{"id":"T59","span":{"begin":2341,"end":2546},"obj":"Sentence"},{"id":"T60","span":{"begin":2547,"end":2660},"obj":"Sentence"},{"id":"T61","span":{"begin":2661,"end":2724},"obj":"Sentence"},{"id":"T62","span":{"begin":2725,"end":2831},"obj":"Sentence"},{"id":"T63","span":{"begin":2832,"end":2918},"obj":"Sentence"},{"id":"T64","span":{"begin":2919,"end":3128},"obj":"Sentence"},{"id":"T65","span":{"begin":3129,"end":3159},"obj":"Sentence"},{"id":"T66","span":{"begin":3160,"end":3329},"obj":"Sentence"},{"id":"T67","span":{"begin":3330,"end":3422},"obj":"Sentence"},{"id":"T68","span":{"begin":3423,"end":3584},"obj":"Sentence"},{"id":"T69","span":{"begin":3585,"end":3621},"obj":"Sentence"},{"id":"T70","span":{"begin":3622,"end":3741},"obj":"Sentence"},{"id":"T71","span":{"begin":3742,"end":3854},"obj":"Sentence"},{"id":"T72","span":{"begin":3855,"end":4008},"obj":"Sentence"},{"id":"T73","span":{"begin":4009,"end":4176},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-PD-MONDO

{"project":"LitCovid-sample-PD-MONDO","denotations":[{"id":"T30","span":{"begin":50,"end":60},"obj":"Disease"},{"id":"T31","span":{"begin":154,"end":164},"obj":"Disease"},{"id":"T32","span":{"begin":2151,"end":2161},"obj":"Disease"},{"id":"T33","span":{"begin":2591,"end":2601},"obj":"Disease"},{"id":"T34","span":{"begin":2678,"end":2688},"obj":"Disease"},{"id":"T35","span":{"begin":3070,"end":3080},"obj":"Disease"}],"attributes":[{"id":"A30","pred":"mondo_id","subj":"T30","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"},{"id":"A31","pred":"mondo_id","subj":"T31","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"},{"id":"A32","pred":"mondo_id","subj":"T32","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"},{"id":"A33","pred":"mondo_id","subj":"T33","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"},{"id":"A34","pred":"mondo_id","subj":"T34","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"},{"id":"A35","pred":"mondo_id","subj":"T35","obj":"http://purl.obolibrary.org/obo/MONDO_0100096"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-UniProt

LitCovid-sample-PD-IDO

{"project":"LitCovid-sample-PD-IDO","denotations":[{"id":"T45","span":{"begin":435,"end":440},"obj":"http://purl.obolibrary.org/obo/CL_0000000"},{"id":"T46","span":{"begin":2386,"end":2390},"obj":"http://purl.obolibrary.org/obo/BFO_0000029"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-PD-FMA

LitCovid-sample-PD-MAT

{"project":"LitCovid-sample-PD-MAT","denotations":[{"id":"T2","span":{"begin":4147,"end":4155},"obj":"http://purl.obolibrary.org/obo/MAT_0000086"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-PD-GO-BP-0

{"project":"LitCovid-sample-PD-GO-BP-0","denotations":[{"id":"T32","span":{"begin":203,"end":216},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T33","span":{"begin":349,"end":362},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T34","span":{"begin":642,"end":652},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T35","span":{"begin":863,"end":873},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T36","span":{"begin":1320,"end":1330},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T37","span":{"begin":1732,"end":1743},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T38","span":{"begin":2357,"end":2379},"obj":"http://purl.obolibrary.org/obo/GO_0006413"},{"id":"T39","span":{"begin":2357,"end":2368},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T40","span":{"begin":2446,"end":2457},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T41","span":{"begin":2855,"end":2868},"obj":"http://purl.obolibrary.org/obo/GO_0070085"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

LitCovid-sample-GO-BP

{"project":"LitCovid-sample-GO-BP","denotations":[{"id":"T30","span":{"begin":203,"end":216},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T31","span":{"begin":349,"end":362},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T32","span":{"begin":642,"end":652},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T33","span":{"begin":863,"end":873},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T34","span":{"begin":1320,"end":1330},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T35","span":{"begin":1732,"end":1743},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T36","span":{"begin":2357,"end":2379},"obj":"http://purl.obolibrary.org/obo/GO_0006413"},{"id":"T37","span":{"begin":2357,"end":2368},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T38","span":{"begin":2446,"end":2457},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T39","span":{"begin":2855,"end":2868},"obj":"http://purl.obolibrary.org/obo/GO_0070085"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

2_test

{"project":"2_test","denotations":[{"id":"32841605-30778233-19659502","span":{"begin":1470,"end":1474},"obj":"30778233"}],"text":"Expression, Purification, and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer and Soluble Human ACE2\nA trimer-stabilized, soluble variant of the SARS-CoV-2 S that contains 22 canonical N-linked glycosylation sequons per protomer and a soluble version of human ACE2 that contains six, lacking the most C-terminal seventh, canonical N-linked glycosylation sequons (Figure 1 A) were purified from the media of transfected HEK293 cells, and the quaternary structure confirmed by negative EM staining for the S trimer (Figure 1B) and purity examined by SDS-PAGE Coomassie G-250 stained gels for both (Figure 1C). In addition, proteolytic digestions followed by proteomic analyses confirmed that the proteins were highly purified (Table S12). Finally, the N terminus of both the mature S and the soluble mature ACE2 were empirically determined via proteolytic digestions and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. These results confirmed that both the secreted, mature forms of S protein and ACE2 begin with an N-terminal glutamine that has undergone condensation to form pyroglutamine at residues 14 and 18, respectively (Figures 1D and S1). The N-terminal peptide observed for S also contains a glycan at Asn-0017 (Figure 1D), and mass spectrometry analysis of non-reducing proteolytic digestions confirmed that Cys-0015 of S is in a disulfide linkage with Cys-0136 (Figure S2; Table S2). Given that SignalP (Almagro Armenteros et al., 2019) predicts signal sequence cleavage between Cys-0015 and Val-0016 but we observed cleavage between Ser-0013 and Gln-0014, we examined the possibility that an in-frame upstream methionine to the proposed start methionine (Figure 1A) might be used to initiate translation (Figure S3). If one examines the predicted signal sequence cleavage using the in-frame Met that is encoded nine amino acids upstream, SignalP now predicts cleavage between the Ser and Gln that we observed in our studies (Figure S3). To examine whether this impacted S expression, we expressed constructs that contained or did not contain the upstream 27 nucleotides in a pseudovirus (VSV) system expressing SARS-CoV-2 S (Figure S4) and in our HEK293 system (data not shown). Both expression systems produced a similar amount of S regardless of which expression construct was utilized (Figure S4). Thus, while the translation initiation start site has still not been fully defined, allowing for earlier translation in expression construct design did not have a significant impact on the generation of S.\nFigure 1 Expression and Characterization of SARS-CoV-2 Spike Glycoprotein Trimer Immunogen and Soluble Human ACE2\n(A) Sequences of SARS-CoV-2 S immunogen and soluble human ACE2. The N-terminal pyroglutamines for both mature protein monomers are bolded, underlined, and shown in green. The canonical N-linked glycosylation sequons are bolded, underlined, and shown in red.\n(B and C) Negative stain electron microscopy of the purified trimer (B) and Coomassie G-250-stained reducing SDS-PAGE gels (C) confirmed purity of the SARS-CoV-2 S protein trimer and of the soluble human ACE2. MWM, molecular weight markers.\n(D) A representative Step-HCD fragmentation spectrum from mass-spectrometry analysis of a tryptic digest of S annotated manually based on search results from pGlyco 2.2. This spectrum defines the N terminus of the mature protein monomer as (pyro-)glutamine 0014. A representative N-glycan consistent with this annotation and our glycomics data (Figure 2) is overlaid by using the Symbol Nomenclature For Glycans (SNFG) code. This complex glycan occurs at N0017. Note, that as expected, the cysteine is carbamidomethylated, and the mass accuracy of the assigned peptide is 0.98 ppm. On the sequence of the N-terminal peptide and in the spectrum, the assigned b (blue) and y (red) ions are shown. In the spectrum, purple highlights glycan oxonium ions and green marks intact peptide fragment ions with various partial glycan sequences still attached. Note that the green-labeled ions allow for limited topology to be extracted including defining that the fucose is on the core and not the antennae of the glycopeptide."}

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