PMC:5118421 / 28517-35809 JSONTXT

{"project":"TEST0","denotations":[{"id":"27920779-179-184-3100255","span":{"begin":501,"end":502},"obj":"[\"20805563\"]"},{"id":"27920779-182-187-3100256","span":{"begin":504,"end":505},"obj":"[\"8011289\"]"},{"id":"27920779-193-199-3100257","span":{"begin":851,"end":853},"obj":"[\"7651532\"]"},{"id":"27920779-229-235-3100258","span":{"begin":1848,"end":1850},"obj":"[\"11859119\"]"},{"id":"27920779-233-239-3100259","span":{"begin":2188,"end":2190},"obj":"[\"15860590\", \"20882016\", \"23940276\"]"},{"id":"27920779-230-236-3100260","span":{"begin":2195,"end":2197},"obj":"[\"23359068\", \"20018688\", \"21908739\", \"23825311\", \"25355869\", \"24614107\", \"23552890\"]"},{"id":"27920779-170-176-3100261","span":{"begin":3173,"end":3175},"obj":"[\"11859119\"]"},{"id":"27920779-84-90-3100262","span":{"begin":3914,"end":3916},"obj":"[\"22407974\"]"},{"id":"27920779-197-203-3100263","span":{"begin":4446,"end":4448},"obj":"[\"22407974\"]"}],"text":"Discussion\nOur study is rooted in the observation that germline-encoded IgV-region genes have inordinately high frequencies of AGY Ser codons, particularly in CDRs. This applies across mouse and human VH, Vκ, and Vλ germline genes but not to αβTCR genes. We show that this trend is conserved even in cartilaginous fishes.\nAGY Ser codons are potentially dangerous because they easily mutate to generate Arg codons with an associated potential to impart antinuclear activity to the respective antibody (1, 3). This raises a paradox because TCN Ser codons do not have this propensity and yet are far less abundant in Ab V region genes and specifically in CDRs. Wagner and colleagues originally hypothesized that this bias toward AGY Ser codons in CDRs was due to a selection pressure to constrain AID motifs to Ag-binding regions of the B cell receptor (17). While this is plausible, our data reveal that this explanation alone cannot account for CDR AGY codon abundance because CDR AGY triplets occur predominantly in the Ser reading frame, even though AID is blind to the translational reading frame. Because TCA and TCG can mutate to stop codons by single-base changes, it is plausible that high CDR AGY/TCN ratios are due in part to selection against these codons. This may hold for TCG which had a low observed/expected ratio, but apparently not for TCA, which had an observed/expected ratio of greater than one, even though it can mutate to a stop codon by two different single-base changes. Overall the observed/expected ratios for TCN codons were greater than one in CDRs. Finally, if there was selection pressure against TCN due to the stop codon potential, we would expect that TCN would be underrepresented in CDRs relative to FRs because there is a bias for increased mutation in CDRs that cannot be explained solely by triplet sequences (13). However no such bias was seen for the Vκ genes of either species (Figure 1A).\nIn view of reports that a measure of autoreactivity may be beneficial in the context of some antiviral antibody responses, we asked whether somatic mutations that generate Arg codons arise frequently in antiviral antibodies, and specifically at AGY codons (37–39, 42–48). While it was not possible to clearly address this question in the context of broadly neutralizing anti-HIV antibodies, we were able to address it in the context of Abs directed against six different viruses. In every case, mutations producing Arg codons were present, often in abundance, and predominantly at AGY codons.\nThis result alone, however, did not provide insight regarding the potential value of antinuclear activity generated via SHM. Our analyses of X-ray structures of Ag–Ab complexes also did not shed light on this question because we examined complexes involving only protein Ags. However, our sequence analyses of antiviral antibodies did reveal a considerable variation in the relative frequency with which an AGY codon mutated to encode an Arg codon versus a codon for Asn or Thr. Based on triplet mutability indices and base preference targeting by AID, we would expect a ~2:1 ratio favoring mutations to Asn/Thr codons over mutations to Arg codons (13). Overall, the Asn + Thr/Arg ratio was 2.7:1 among combined antiviral antibodies, suggesting some selection pressure against Arg. However, there was considerable variation among different antiviral antibodies. For example, while the 2:1 ratio closely approximated that seen for antibodies to hepatitis virus, the ratio was ~3.5:1 for antibodies against influenza. It is unclear whether deviations from the expected ratio are due to the autoreactive properties of CDR Arg residues or simply due to direct Ag-contact considerations. Arg is larger than Asn or Thr, such that replacing Ser with Arg may impede Ag engagement more often due to steric effects. Results of our analysis together with those of a prior study by Raghunathan et al. (19), however, indicate that Arg residues in Ab V regions frequently make contact with protein Ags. Thus, regardless of whether Ab affinity for nuclear Ags is beneficial to some viral immune responses, somatic mutations that produce Arg codons at germline CDR AGY codons can be beneficial to the development of high-avidity antibodies.\nWe also found, unexpectedly, that AGY codons in antiviral Abs mutated frequently to codons for most of the other amino acids that were identified as key Ag-contact residues in the Ab-binding site (19). Only a single-base change was required to generate codons for most of these key residues. Among the antiviral Abs we analyzed, point mutations in AGY that generated codons for these key residues occurred predominantly at G and C, which are the major initiation sites for SHM by AID.\nFinally, upon analyzing X-ray structures of immune complexes involving protein Ags, we found that Ag-contact residues created by SHM occurred more frequently in AGY codons than in any other synonymous codon group. And this was also true for the key contact residues defined by Raghunathan and colleagues primarily on the basis of germline-encoded contacts. Notably, all of these key contact residues are polar or charged. Polar and charged amino acids are preferentially found on solvent-exposed surfaces of all proteins. Additionally, small polar amino acids are often favored in loop regions where they contribute both to flexibility and direct contacts with other protein ligands due to small side chains with minimal steric barriers. Polar residues, such as Ser, Asn, and Thr, can act as both hydrogen bond donors and acceptors thus making them ideal residues to accommodate a number of different binding landscapes: they can form hydrogen bonds with other polar residues as well as basic and acidic residues (49, 50). Serine, being one of the smallest amino acids, is perhaps the most compliant residue. Other small amino acids, such as Cys and Ala, would be less favored do to unwanted disulfide bond formation (Cys) or lack of hydrogen bonding (Ala).\nMutation of Ser to another small to midsize polar residue, such as Thr, Gly, and Asn, maintains most of the binding plasticity of serine while potentially adding additional binding energies from either increased van der Waals interactions, stronger hydrogen bond strength due to decreased hydrogen bond length, or both. Thus serine is an ideal residue for contributing to binding on its own, while, at the same time, being an ideal starting point for mutation to other small polar groups. Replacing Ser with a larger amino acid such as Arg during SHM, while beneficial in some cases, may come with a higher probability of disrupting the interaction between Ab and Ag. This may account for the high ratio of Asn and Thr to Arg replacement mutations at CDR AGY codons of influenza antibodies. It is notable that unlike the case for AGY codons, random base substitutions in TCN Ser codons often lead to large hydrophobic residues or to less favorable residues, such as Ala (non-polar) and Cys (potentially disruptive). In sum, the fact that Ser is one of the seven major Ag-contact residues, together with the ease with which AGY Ser codons can mutate to four more of these residues, provides the most straightforward explanation of why AGY codon abundance in Ab CDRs is conserved from sharks to humans."}

{"project":"2_test","denotations":[{"id":"27920779-20805563-34707978","span":{"begin":501,"end":502},"obj":"20805563"},{"id":"27920779-8011289-34707979","span":{"begin":504,"end":505},"obj":"8011289"},{"id":"27920779-7651532-34707980","span":{"begin":851,"end":853},"obj":"7651532"},{"id":"27920779-11859119-34707981","span":{"begin":1848,"end":1850},"obj":"11859119"},{"id":"27920779-15860590-34707982","span":{"begin":2188,"end":2190},"obj":"15860590"},{"id":"27920779-20882016-34707982","span":{"begin":2188,"end":2190},"obj":"20882016"},{"id":"27920779-23940276-34707982","span":{"begin":2188,"end":2190},"obj":"23940276"},{"id":"27920779-23359068-34707983","span":{"begin":2195,"end":2197},"obj":"23359068"},{"id":"27920779-20018688-34707983","span":{"begin":2195,"end":2197},"obj":"20018688"},{"id":"27920779-21908739-34707983","span":{"begin":2195,"end":2197},"obj":"21908739"},{"id":"27920779-23825311-34707983","span":{"begin":2195,"end":2197},"obj":"23825311"},{"id":"27920779-25355869-34707983","span":{"begin":2195,"end":2197},"obj":"25355869"},{"id":"27920779-24614107-34707983","span":{"begin":2195,"end":2197},"obj":"24614107"},{"id":"27920779-23552890-34707983","span":{"begin":2195,"end":2197},"obj":"23552890"},{"id":"27920779-11859119-34707984","span":{"begin":3173,"end":3175},"obj":"11859119"},{"id":"27920779-22407974-34707985","span":{"begin":3914,"end":3916},"obj":"22407974"},{"id":"27920779-22407974-34707986","span":{"begin":4446,"end":4448},"obj":"22407974"}],"text":"Discussion\nOur study is rooted in the observation that germline-encoded IgV-region genes have inordinately high frequencies of AGY Ser codons, particularly in CDRs. This applies across mouse and human VH, Vκ, and Vλ germline genes but not to αβTCR genes. We show that this trend is conserved even in cartilaginous fishes.\nAGY Ser codons are potentially dangerous because they easily mutate to generate Arg codons with an associated potential to impart antinuclear activity to the respective antibody (1, 3). This raises a paradox because TCN Ser codons do not have this propensity and yet are far less abundant in Ab V region genes and specifically in CDRs. Wagner and colleagues originally hypothesized that this bias toward AGY Ser codons in CDRs was due to a selection pressure to constrain AID motifs to Ag-binding regions of the B cell receptor (17). While this is plausible, our data reveal that this explanation alone cannot account for CDR AGY codon abundance because CDR AGY triplets occur predominantly in the Ser reading frame, even though AID is blind to the translational reading frame. Because TCA and TCG can mutate to stop codons by single-base changes, it is plausible that high CDR AGY/TCN ratios are due in part to selection against these codons. This may hold for TCG which had a low observed/expected ratio, but apparently not for TCA, which had an observed/expected ratio of greater than one, even though it can mutate to a stop codon by two different single-base changes. Overall the observed/expected ratios for TCN codons were greater than one in CDRs. Finally, if there was selection pressure against TCN due to the stop codon potential, we would expect that TCN would be underrepresented in CDRs relative to FRs because there is a bias for increased mutation in CDRs that cannot be explained solely by triplet sequences (13). However no such bias was seen for the Vκ genes of either species (Figure 1A).\nIn view of reports that a measure of autoreactivity may be beneficial in the context of some antiviral antibody responses, we asked whether somatic mutations that generate Arg codons arise frequently in antiviral antibodies, and specifically at AGY codons (37–39, 42–48). While it was not possible to clearly address this question in the context of broadly neutralizing anti-HIV antibodies, we were able to address it in the context of Abs directed against six different viruses. In every case, mutations producing Arg codons were present, often in abundance, and predominantly at AGY codons.\nThis result alone, however, did not provide insight regarding the potential value of antinuclear activity generated via SHM. Our analyses of X-ray structures of Ag–Ab complexes also did not shed light on this question because we examined complexes involving only protein Ags. However, our sequence analyses of antiviral antibodies did reveal a considerable variation in the relative frequency with which an AGY codon mutated to encode an Arg codon versus a codon for Asn or Thr. Based on triplet mutability indices and base preference targeting by AID, we would expect a ~2:1 ratio favoring mutations to Asn/Thr codons over mutations to Arg codons (13). Overall, the Asn + Thr/Arg ratio was 2.7:1 among combined antiviral antibodies, suggesting some selection pressure against Arg. However, there was considerable variation among different antiviral antibodies. For example, while the 2:1 ratio closely approximated that seen for antibodies to hepatitis virus, the ratio was ~3.5:1 for antibodies against influenza. It is unclear whether deviations from the expected ratio are due to the autoreactive properties of CDR Arg residues or simply due to direct Ag-contact considerations. Arg is larger than Asn or Thr, such that replacing Ser with Arg may impede Ag engagement more often due to steric effects. Results of our analysis together with those of a prior study by Raghunathan et al. (19), however, indicate that Arg residues in Ab V regions frequently make contact with protein Ags. Thus, regardless of whether Ab affinity for nuclear Ags is beneficial to some viral immune responses, somatic mutations that produce Arg codons at germline CDR AGY codons can be beneficial to the development of high-avidity antibodies.\nWe also found, unexpectedly, that AGY codons in antiviral Abs mutated frequently to codons for most of the other amino acids that were identified as key Ag-contact residues in the Ab-binding site (19). Only a single-base change was required to generate codons for most of these key residues. Among the antiviral Abs we analyzed, point mutations in AGY that generated codons for these key residues occurred predominantly at G and C, which are the major initiation sites for SHM by AID.\nFinally, upon analyzing X-ray structures of immune complexes involving protein Ags, we found that Ag-contact residues created by SHM occurred more frequently in AGY codons than in any other synonymous codon group. And this was also true for the key contact residues defined by Raghunathan and colleagues primarily on the basis of germline-encoded contacts. Notably, all of these key contact residues are polar or charged. Polar and charged amino acids are preferentially found on solvent-exposed surfaces of all proteins. Additionally, small polar amino acids are often favored in loop regions where they contribute both to flexibility and direct contacts with other protein ligands due to small side chains with minimal steric barriers. Polar residues, such as Ser, Asn, and Thr, can act as both hydrogen bond donors and acceptors thus making them ideal residues to accommodate a number of different binding landscapes: they can form hydrogen bonds with other polar residues as well as basic and acidic residues (49, 50). Serine, being one of the smallest amino acids, is perhaps the most compliant residue. Other small amino acids, such as Cys and Ala, would be less favored do to unwanted disulfide bond formation (Cys) or lack of hydrogen bonding (Ala).\nMutation of Ser to another small to midsize polar residue, such as Thr, Gly, and Asn, maintains most of the binding plasticity of serine while potentially adding additional binding energies from either increased van der Waals interactions, stronger hydrogen bond strength due to decreased hydrogen bond length, or both. Thus serine is an ideal residue for contributing to binding on its own, while, at the same time, being an ideal starting point for mutation to other small polar groups. Replacing Ser with a larger amino acid such as Arg during SHM, while beneficial in some cases, may come with a higher probability of disrupting the interaction between Ab and Ag. This may account for the high ratio of Asn and Thr to Arg replacement mutations at CDR AGY codons of influenza antibodies. It is notable that unlike the case for AGY codons, random base substitutions in TCN Ser codons often lead to large hydrophobic residues or to less favorable residues, such as Ala (non-polar) and Cys (potentially disruptive). In sum, the fact that Ser is one of the seven major Ag-contact residues, together with the ease with which AGY Ser codons can mutate to four more of these residues, provides the most straightforward explanation of why AGY codon abundance in Ab CDRs is conserved from sharks to humans."}

{"project":"MyTest","denotations":[{"id":"27920779-20805563-34707978","span":{"begin":501,"end":502},"obj":"20805563"},{"id":"27920779-8011289-34707979","span":{"begin":504,"end":505},"obj":"8011289"},{"id":"27920779-7651532-34707980","span":{"begin":851,"end":853},"obj":"7651532"},{"id":"27920779-11859119-34707981","span":{"begin":1848,"end":1850},"obj":"11859119"},{"id":"27920779-15860590-34707982","span":{"begin":2188,"end":2190},"obj":"15860590"},{"id":"27920779-20882016-34707982","span":{"begin":2188,"end":2190},"obj":"20882016"},{"id":"27920779-23940276-34707982","span":{"begin":2188,"end":2190},"obj":"23940276"},{"id":"27920779-23359068-34707983","span":{"begin":2195,"end":2197},"obj":"23359068"},{"id":"27920779-20018688-34707983","span":{"begin":2195,"end":2197},"obj":"20018688"},{"id":"27920779-21908739-34707983","span":{"begin":2195,"end":2197},"obj":"21908739"},{"id":"27920779-23825311-34707983","span":{"begin":2195,"end":2197},"obj":"23825311"},{"id":"27920779-25355869-34707983","span":{"begin":2195,"end":2197},"obj":"25355869"},{"id":"27920779-24614107-34707983","span":{"begin":2195,"end":2197},"obj":"24614107"},{"id":"27920779-23552890-34707983","span":{"begin":2195,"end":2197},"obj":"23552890"},{"id":"27920779-11859119-34707984","span":{"begin":3173,"end":3175},"obj":"11859119"},{"id":"27920779-22407974-34707985","span":{"begin":3914,"end":3916},"obj":"22407974"},{"id":"27920779-22407974-34707986","span":{"begin":4446,"end":4448},"obj":"22407974"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Discussion\nOur study is rooted in the observation that germline-encoded IgV-region genes have inordinately high frequencies of AGY Ser codons, particularly in CDRs. This applies across mouse and human VH, Vκ, and Vλ germline genes but not to αβTCR genes. We show that this trend is conserved even in cartilaginous fishes.\nAGY Ser codons are potentially dangerous because they easily mutate to generate Arg codons with an associated potential to impart antinuclear activity to the respective antibody (1, 3). This raises a paradox because TCN Ser codons do not have this propensity and yet are far less abundant in Ab V region genes and specifically in CDRs. Wagner and colleagues originally hypothesized that this bias toward AGY Ser codons in CDRs was due to a selection pressure to constrain AID motifs to Ag-binding regions of the B cell receptor (17). While this is plausible, our data reveal that this explanation alone cannot account for CDR AGY codon abundance because CDR AGY triplets occur predominantly in the Ser reading frame, even though AID is blind to the translational reading frame. Because TCA and TCG can mutate to stop codons by single-base changes, it is plausible that high CDR AGY/TCN ratios are due in part to selection against these codons. This may hold for TCG which had a low observed/expected ratio, but apparently not for TCA, which had an observed/expected ratio of greater than one, even though it can mutate to a stop codon by two different single-base changes. Overall the observed/expected ratios for TCN codons were greater than one in CDRs. Finally, if there was selection pressure against TCN due to the stop codon potential, we would expect that TCN would be underrepresented in CDRs relative to FRs because there is a bias for increased mutation in CDRs that cannot be explained solely by triplet sequences (13). However no such bias was seen for the Vκ genes of either species (Figure 1A).\nIn view of reports that a measure of autoreactivity may be beneficial in the context of some antiviral antibody responses, we asked whether somatic mutations that generate Arg codons arise frequently in antiviral antibodies, and specifically at AGY codons (37–39, 42–48). While it was not possible to clearly address this question in the context of broadly neutralizing anti-HIV antibodies, we were able to address it in the context of Abs directed against six different viruses. In every case, mutations producing Arg codons were present, often in abundance, and predominantly at AGY codons.\nThis result alone, however, did not provide insight regarding the potential value of antinuclear activity generated via SHM. Our analyses of X-ray structures of Ag–Ab complexes also did not shed light on this question because we examined complexes involving only protein Ags. However, our sequence analyses of antiviral antibodies did reveal a considerable variation in the relative frequency with which an AGY codon mutated to encode an Arg codon versus a codon for Asn or Thr. Based on triplet mutability indices and base preference targeting by AID, we would expect a ~2:1 ratio favoring mutations to Asn/Thr codons over mutations to Arg codons (13). Overall, the Asn + Thr/Arg ratio was 2.7:1 among combined antiviral antibodies, suggesting some selection pressure against Arg. However, there was considerable variation among different antiviral antibodies. For example, while the 2:1 ratio closely approximated that seen for antibodies to hepatitis virus, the ratio was ~3.5:1 for antibodies against influenza. It is unclear whether deviations from the expected ratio are due to the autoreactive properties of CDR Arg residues or simply due to direct Ag-contact considerations. Arg is larger than Asn or Thr, such that replacing Ser with Arg may impede Ag engagement more often due to steric effects. Results of our analysis together with those of a prior study by Raghunathan et al. (19), however, indicate that Arg residues in Ab V regions frequently make contact with protein Ags. Thus, regardless of whether Ab affinity for nuclear Ags is beneficial to some viral immune responses, somatic mutations that produce Arg codons at germline CDR AGY codons can be beneficial to the development of high-avidity antibodies.\nWe also found, unexpectedly, that AGY codons in antiviral Abs mutated frequently to codons for most of the other amino acids that were identified as key Ag-contact residues in the Ab-binding site (19). Only a single-base change was required to generate codons for most of these key residues. Among the antiviral Abs we analyzed, point mutations in AGY that generated codons for these key residues occurred predominantly at G and C, which are the major initiation sites for SHM by AID.\nFinally, upon analyzing X-ray structures of immune complexes involving protein Ags, we found that Ag-contact residues created by SHM occurred more frequently in AGY codons than in any other synonymous codon group. And this was also true for the key contact residues defined by Raghunathan and colleagues primarily on the basis of germline-encoded contacts. Notably, all of these key contact residues are polar or charged. Polar and charged amino acids are preferentially found on solvent-exposed surfaces of all proteins. Additionally, small polar amino acids are often favored in loop regions where they contribute both to flexibility and direct contacts with other protein ligands due to small side chains with minimal steric barriers. Polar residues, such as Ser, Asn, and Thr, can act as both hydrogen bond donors and acceptors thus making them ideal residues to accommodate a number of different binding landscapes: they can form hydrogen bonds with other polar residues as well as basic and acidic residues (49, 50). Serine, being one of the smallest amino acids, is perhaps the most compliant residue. Other small amino acids, such as Cys and Ala, would be less favored do to unwanted disulfide bond formation (Cys) or lack of hydrogen bonding (Ala).\nMutation of Ser to another small to midsize polar residue, such as Thr, Gly, and Asn, maintains most of the binding plasticity of serine while potentially adding additional binding energies from either increased van der Waals interactions, stronger hydrogen bond strength due to decreased hydrogen bond length, or both. Thus serine is an ideal residue for contributing to binding on its own, while, at the same time, being an ideal starting point for mutation to other small polar groups. Replacing Ser with a larger amino acid such as Arg during SHM, while beneficial in some cases, may come with a higher probability of disrupting the interaction between Ab and Ag. This may account for the high ratio of Asn and Thr to Arg replacement mutations at CDR AGY codons of influenza antibodies. It is notable that unlike the case for AGY codons, random base substitutions in TCN Ser codons often lead to large hydrophobic residues or to less favorable residues, such as Ala (non-polar) and Cys (potentially disruptive). In sum, the fact that Ser is one of the seven major Ag-contact residues, together with the ease with which AGY Ser codons can mutate to four more of these residues, provides the most straightforward explanation of why AGY codon abundance in Ab CDRs is conserved from sharks to humans."}