PMC:5118421 / 22414-28515 JSONTXT

{"project":"TEST0","denotations":[{"id":"27920779-183-189-3100246","span":{"begin":458,"end":460},"obj":"[\"11859119\"]"},{"id":"27920779-144-150-3100247","span":{"begin":882,"end":884},"obj":"[\"22407974\"]"},{"id":"27920779-56-62-3100248","span":{"begin":2143,"end":2145},"obj":"[\"22407974\"]"},{"id":"27920779-125-131-3100249","span":{"begin":3426,"end":3428},"obj":"[\"22407974\"]"},{"id":"27920779-62-68-3100250","span":{"begin":4826,"end":4828},"obj":"[\"11859119\"]"},{"id":"27920779-66-72-3100251","span":{"begin":4830,"end":4832},"obj":"[\"12943801\"]"},{"id":"27920779-70-76-3100252","span":{"begin":4834,"end":4836},"obj":"[\"8786330\"]"},{"id":"27920779-237-243-3100253","span":{"begin":5368,"end":5370},"obj":"[\"7916950\"]"},{"id":"27920779-86-92-3100254","span":{"begin":5990,"end":5992},"obj":"[\"22407974\"]"}],"text":"CDR AGY Codons Frequently Mutate to Produce Codons for Key Ag-Contact Residues in the Ab-Binding Site\nOur analyses of somatic mutations in antiviral Ab led to an unexpected finding: CDR AGY Ser codons frequently mutated to Asn, Thr, and Gly codons in addition to Arg codons. Most of these mutations occurred by single-base changes, predominantly at the central base in the AGY triplet (Table 2), which is the position that is preferentially targeted by AID (13). In many cases, mutations to these alternative codons, particularly those for Asn and Thr, were more frequent than to Arg codons. For example, in anti-influenza Abs, CDR AGY mutations to Asn and Thr codons were each approximately twice as frequent as mutations to Arg codons. These observations were particularly revealing because in their analyses of numerous crystal structures of Ab–Ag complexes, Raghunathan et al. (19) identified Asn, Thr, Arg, Gly, Ser, Asp, and Tyr as key (i.e., most frequent) Ag-contact residues.\nTable 2 Base distribution of somatic mutations in CDR AGY Ser codons.\nImmunogen AGY (%) AGY (%) AGY (%) 2 changes (%) 3 changes (%)\nInfluenzaa 12 53 11 20 4\nInfluenzab 11 52 15 20 2\nWest Nile 0 80 20 0 0\nDengue 15 57 14 14 0\nRhinovirus 0 22 19 52 7\nAvian Influenza 0 67 33 0 0\nHep. A, B, and C 12 35 18 35 0\naAntibody sequences from Wrammert et al. (23).\nbAntibody sequences from Li et al. (24). In the report by Raghunathan and colleagues, it was not clear which contact residues were generated by SHM. To determine if residues frequently generated by SHM of AGY Ser codons are associated with Ab affinity maturation, we analyzed 72 (46 mouse and 26 human) Ab–Ag crystal structures available in the RCSB protein data bank (pdb) database, identified predicted Ag-contact residues, and searched IgBLAST to distinguish those that were germline-encoded from those that were somatically generated. When mouse and human data where combined, the seven most frequent Ag-contact residues were Arg, Asp, Asn, Gly, Ser, Thr and Tyr (Figure S4 in Supplementary Material). This result is identical to that of Raghunathan et al. (19), even though only 4 of the 72 structures we analyzed were also analyzed by them. Yet, we found that only three (Asn, Ser, and Tyr) of those seven residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr) were present at higher frequencies than expected within CDRs of mouse and human germline IgV-region genes (Figure 4A). Importantly, amino acids resulting from SHM accounted for only 10–23% (average 14.7%) of all Ag-contact residues (Table 3 footnotes; Figure S4 in Supplementary Material). This is relevant to our conclusion regarding AGY versatility because it means that the seven key Ag-contact residues were largely defined by germline-encoded contacts; yet four (Asn, Arg, Gly, and Thr) of the seven most abundant contact residues arise frequently from somatic mutations at CDR AGY codons.\nFigure 4 CDR AGY Ser codons play a key role in affinity maturation. (A) Ratio observed over expected for synonymous codons in CDR sequences of combined IgV genes (VH, Vκ, and Vλ). (B) Percentage of the total contact residues that were created by SHM in V-region sequences only. Each data set represents a germline-encoded codon given rise to any contact residue. Black bars represent the percentage of AGY Ser codons that gave rise to a key contact residue defined by Raghunathan et al. (19).\nTable 3 Amino acid replacements due to somatic mutation of germline AGY Ser codons.a\nContact mutations at AGYb % of all contact mutationsc\nHuman Mouse Human (%) Mouse (%)\nArg 4 7 5.55 6.73\nAsn 5 9 6.94 8.65\nGly 0 1 0 0.96\nThr 6 5 8.33 4.81\nOthers 15 8 20.83 7.69\naData from 26 human and 46 mouse crystal structures of Ag–Ab complexes.\nbV-region contact residues arising from SHM of AGY Ser codons. Numbers expressed in absolute numbers. Total contact residues analyzed were 317 (human) and 886 (mouse). Total contact residues that were associated with SHM of a V-region codon were 72 (human) and 104 (mouse).\ncPercentage of total somatically generated contacts residues that arose from mutation of AGY Ser codons. For somatically generated contact residues, mutations at AGY Ser codons were the most abundant by far, and occurred ~2–3 times more often than mutations at AAY Asn codons (Figure 4B), the second most consistently mutated codon group. Most importantly, AGY Ser codons mutated to contact residues more often than any other codon group (Figure 4B), and a large proportion of these (~70%) were those defined as key Ag-contact residues. AGY mutations to codons for Arg, Asn, and Thr were the most consistent, and this was true for both contact and non-contact residues (Table 3 and data not shown). AAY triplets are also intrinsically preferred targets of SHM (13, 15, 16). However, when considering the potential to mutate to 1 of the 6 non-synonymous key contact residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr), AGY Ser codons are able to do so via 12 out of 18 possible single-base changes. For AAY (Asn), this occurs with 8 out of 18 base changes, and for TCN, it occurs with only 6 out of 36 base substitutions (Figure 5), a result that is in agreement with the observation by Chang and Casali that CDR, but not FR sequences, are prone to acquire replacement mutations upon random point mutation (41). Collectively, the results of these analyses indicate that AGY codons contribute to Ab affinity both directly, by encoding a Ser residue, and indirectly due to the ease with which they mutate to encode other residues beneficial to the process of Ab affinity maturation. We believe this is the most straightforward explanation for the conservation of AGY codon abundance in CDRs of germline IgV-region genes.\nFigure 5 AGY Ser codons plasticity. Probability of creating a key non-synonymous contact residue by one nucleotide change. Filled gray boxes indicate a key Ag-contact residue as defined by Raghunathan et al. (19). White boxes indicate a synonymous change, a non-key contact residue (defined in the text) or a stop codon."}

{"project":"2_test","denotations":[{"id":"27920779-11859119-34707969","span":{"begin":458,"end":460},"obj":"11859119"},{"id":"27920779-22407974-34707970","span":{"begin":882,"end":884},"obj":"22407974"},{"id":"27920779-22407974-34707971","span":{"begin":2143,"end":2145},"obj":"22407974"},{"id":"27920779-22407974-34707972","span":{"begin":3426,"end":3428},"obj":"22407974"},{"id":"27920779-11859119-34707973","span":{"begin":4826,"end":4828},"obj":"11859119"},{"id":"27920779-12943801-34707974","span":{"begin":4830,"end":4832},"obj":"12943801"},{"id":"27920779-8786330-34707975","span":{"begin":4834,"end":4836},"obj":"8786330"},{"id":"27920779-7916950-34707976","span":{"begin":5368,"end":5370},"obj":"7916950"},{"id":"27920779-22407974-34707977","span":{"begin":5990,"end":5992},"obj":"22407974"}],"text":"CDR AGY Codons Frequently Mutate to Produce Codons for Key Ag-Contact Residues in the Ab-Binding Site\nOur analyses of somatic mutations in antiviral Ab led to an unexpected finding: CDR AGY Ser codons frequently mutated to Asn, Thr, and Gly codons in addition to Arg codons. Most of these mutations occurred by single-base changes, predominantly at the central base in the AGY triplet (Table 2), which is the position that is preferentially targeted by AID (13). In many cases, mutations to these alternative codons, particularly those for Asn and Thr, were more frequent than to Arg codons. For example, in anti-influenza Abs, CDR AGY mutations to Asn and Thr codons were each approximately twice as frequent as mutations to Arg codons. These observations were particularly revealing because in their analyses of numerous crystal structures of Ab–Ag complexes, Raghunathan et al. (19) identified Asn, Thr, Arg, Gly, Ser, Asp, and Tyr as key (i.e., most frequent) Ag-contact residues.\nTable 2 Base distribution of somatic mutations in CDR AGY Ser codons.\nImmunogen AGY (%) AGY (%) AGY (%) 2 changes (%) 3 changes (%)\nInfluenzaa 12 53 11 20 4\nInfluenzab 11 52 15 20 2\nWest Nile 0 80 20 0 0\nDengue 15 57 14 14 0\nRhinovirus 0 22 19 52 7\nAvian Influenza 0 67 33 0 0\nHep. A, B, and C 12 35 18 35 0\naAntibody sequences from Wrammert et al. (23).\nbAntibody sequences from Li et al. (24). In the report by Raghunathan and colleagues, it was not clear which contact residues were generated by SHM. To determine if residues frequently generated by SHM of AGY Ser codons are associated with Ab affinity maturation, we analyzed 72 (46 mouse and 26 human) Ab–Ag crystal structures available in the RCSB protein data bank (pdb) database, identified predicted Ag-contact residues, and searched IgBLAST to distinguish those that were germline-encoded from those that were somatically generated. When mouse and human data where combined, the seven most frequent Ag-contact residues were Arg, Asp, Asn, Gly, Ser, Thr and Tyr (Figure S4 in Supplementary Material). This result is identical to that of Raghunathan et al. (19), even though only 4 of the 72 structures we analyzed were also analyzed by them. Yet, we found that only three (Asn, Ser, and Tyr) of those seven residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr) were present at higher frequencies than expected within CDRs of mouse and human germline IgV-region genes (Figure 4A). Importantly, amino acids resulting from SHM accounted for only 10–23% (average 14.7%) of all Ag-contact residues (Table 3 footnotes; Figure S4 in Supplementary Material). This is relevant to our conclusion regarding AGY versatility because it means that the seven key Ag-contact residues were largely defined by germline-encoded contacts; yet four (Asn, Arg, Gly, and Thr) of the seven most abundant contact residues arise frequently from somatic mutations at CDR AGY codons.\nFigure 4 CDR AGY Ser codons play a key role in affinity maturation. (A) Ratio observed over expected for synonymous codons in CDR sequences of combined IgV genes (VH, Vκ, and Vλ). (B) Percentage of the total contact residues that were created by SHM in V-region sequences only. Each data set represents a germline-encoded codon given rise to any contact residue. Black bars represent the percentage of AGY Ser codons that gave rise to a key contact residue defined by Raghunathan et al. (19).\nTable 3 Amino acid replacements due to somatic mutation of germline AGY Ser codons.a\nContact mutations at AGYb % of all contact mutationsc\nHuman Mouse Human (%) Mouse (%)\nArg 4 7 5.55 6.73\nAsn 5 9 6.94 8.65\nGly 0 1 0 0.96\nThr 6 5 8.33 4.81\nOthers 15 8 20.83 7.69\naData from 26 human and 46 mouse crystal structures of Ag–Ab complexes.\nbV-region contact residues arising from SHM of AGY Ser codons. Numbers expressed in absolute numbers. Total contact residues analyzed were 317 (human) and 886 (mouse). Total contact residues that were associated with SHM of a V-region codon were 72 (human) and 104 (mouse).\ncPercentage of total somatically generated contacts residues that arose from mutation of AGY Ser codons. For somatically generated contact residues, mutations at AGY Ser codons were the most abundant by far, and occurred ~2–3 times more often than mutations at AAY Asn codons (Figure 4B), the second most consistently mutated codon group. Most importantly, AGY Ser codons mutated to contact residues more often than any other codon group (Figure 4B), and a large proportion of these (~70%) were those defined as key Ag-contact residues. AGY mutations to codons for Arg, Asn, and Thr were the most consistent, and this was true for both contact and non-contact residues (Table 3 and data not shown). AAY triplets are also intrinsically preferred targets of SHM (13, 15, 16). However, when considering the potential to mutate to 1 of the 6 non-synonymous key contact residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr), AGY Ser codons are able to do so via 12 out of 18 possible single-base changes. For AAY (Asn), this occurs with 8 out of 18 base changes, and for TCN, it occurs with only 6 out of 36 base substitutions (Figure 5), a result that is in agreement with the observation by Chang and Casali that CDR, but not FR sequences, are prone to acquire replacement mutations upon random point mutation (41). Collectively, the results of these analyses indicate that AGY codons contribute to Ab affinity both directly, by encoding a Ser residue, and indirectly due to the ease with which they mutate to encode other residues beneficial to the process of Ab affinity maturation. We believe this is the most straightforward explanation for the conservation of AGY codon abundance in CDRs of germline IgV-region genes.\nFigure 5 AGY Ser codons plasticity. Probability of creating a key non-synonymous contact residue by one nucleotide change. Filled gray boxes indicate a key Ag-contact residue as defined by Raghunathan et al. (19). White boxes indicate a synonymous change, a non-key contact residue (defined in the text) or a stop codon."}

{"project":"MyTest","denotations":[{"id":"27920779-11859119-34707969","span":{"begin":458,"end":460},"obj":"11859119"},{"id":"27920779-22407974-34707970","span":{"begin":882,"end":884},"obj":"22407974"},{"id":"27920779-22407974-34707971","span":{"begin":2143,"end":2145},"obj":"22407974"},{"id":"27920779-22407974-34707972","span":{"begin":3426,"end":3428},"obj":"22407974"},{"id":"27920779-11859119-34707973","span":{"begin":4826,"end":4828},"obj":"11859119"},{"id":"27920779-12943801-34707974","span":{"begin":4830,"end":4832},"obj":"12943801"},{"id":"27920779-8786330-34707975","span":{"begin":4834,"end":4836},"obj":"8786330"},{"id":"27920779-7916950-34707976","span":{"begin":5368,"end":5370},"obj":"7916950"},{"id":"27920779-22407974-34707977","span":{"begin":5990,"end":5992},"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":"CDR AGY Codons Frequently Mutate to Produce Codons for Key Ag-Contact Residues in the Ab-Binding Site\nOur analyses of somatic mutations in antiviral Ab led to an unexpected finding: CDR AGY Ser codons frequently mutated to Asn, Thr, and Gly codons in addition to Arg codons. Most of these mutations occurred by single-base changes, predominantly at the central base in the AGY triplet (Table 2), which is the position that is preferentially targeted by AID (13). In many cases, mutations to these alternative codons, particularly those for Asn and Thr, were more frequent than to Arg codons. For example, in anti-influenza Abs, CDR AGY mutations to Asn and Thr codons were each approximately twice as frequent as mutations to Arg codons. These observations were particularly revealing because in their analyses of numerous crystal structures of Ab–Ag complexes, Raghunathan et al. (19) identified Asn, Thr, Arg, Gly, Ser, Asp, and Tyr as key (i.e., most frequent) Ag-contact residues.\nTable 2 Base distribution of somatic mutations in CDR AGY Ser codons.\nImmunogen AGY (%) AGY (%) AGY (%) 2 changes (%) 3 changes (%)\nInfluenzaa 12 53 11 20 4\nInfluenzab 11 52 15 20 2\nWest Nile 0 80 20 0 0\nDengue 15 57 14 14 0\nRhinovirus 0 22 19 52 7\nAvian Influenza 0 67 33 0 0\nHep. A, B, and C 12 35 18 35 0\naAntibody sequences from Wrammert et al. (23).\nbAntibody sequences from Li et al. (24). In the report by Raghunathan and colleagues, it was not clear which contact residues were generated by SHM. To determine if residues frequently generated by SHM of AGY Ser codons are associated with Ab affinity maturation, we analyzed 72 (46 mouse and 26 human) Ab–Ag crystal structures available in the RCSB protein data bank (pdb) database, identified predicted Ag-contact residues, and searched IgBLAST to distinguish those that were germline-encoded from those that were somatically generated. When mouse and human data where combined, the seven most frequent Ag-contact residues were Arg, Asp, Asn, Gly, Ser, Thr and Tyr (Figure S4 in Supplementary Material). This result is identical to that of Raghunathan et al. (19), even though only 4 of the 72 structures we analyzed were also analyzed by them. Yet, we found that only three (Asn, Ser, and Tyr) of those seven residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr) were present at higher frequencies than expected within CDRs of mouse and human germline IgV-region genes (Figure 4A). Importantly, amino acids resulting from SHM accounted for only 10–23% (average 14.7%) of all Ag-contact residues (Table 3 footnotes; Figure S4 in Supplementary Material). This is relevant to our conclusion regarding AGY versatility because it means that the seven key Ag-contact residues were largely defined by germline-encoded contacts; yet four (Asn, Arg, Gly, and Thr) of the seven most abundant contact residues arise frequently from somatic mutations at CDR AGY codons.\nFigure 4 CDR AGY Ser codons play a key role in affinity maturation. (A) Ratio observed over expected for synonymous codons in CDR sequences of combined IgV genes (VH, Vκ, and Vλ). (B) Percentage of the total contact residues that were created by SHM in V-region sequences only. Each data set represents a germline-encoded codon given rise to any contact residue. Black bars represent the percentage of AGY Ser codons that gave rise to a key contact residue defined by Raghunathan et al. (19).\nTable 3 Amino acid replacements due to somatic mutation of germline AGY Ser codons.a\nContact mutations at AGYb % of all contact mutationsc\nHuman Mouse Human (%) Mouse (%)\nArg 4 7 5.55 6.73\nAsn 5 9 6.94 8.65\nGly 0 1 0 0.96\nThr 6 5 8.33 4.81\nOthers 15 8 20.83 7.69\naData from 26 human and 46 mouse crystal structures of Ag–Ab complexes.\nbV-region contact residues arising from SHM of AGY Ser codons. Numbers expressed in absolute numbers. Total contact residues analyzed were 317 (human) and 886 (mouse). Total contact residues that were associated with SHM of a V-region codon were 72 (human) and 104 (mouse).\ncPercentage of total somatically generated contacts residues that arose from mutation of AGY Ser codons. For somatically generated contact residues, mutations at AGY Ser codons were the most abundant by far, and occurred ~2–3 times more often than mutations at AAY Asn codons (Figure 4B), the second most consistently mutated codon group. Most importantly, AGY Ser codons mutated to contact residues more often than any other codon group (Figure 4B), and a large proportion of these (~70%) were those defined as key Ag-contact residues. AGY mutations to codons for Arg, Asn, and Thr were the most consistent, and this was true for both contact and non-contact residues (Table 3 and data not shown). AAY triplets are also intrinsically preferred targets of SHM (13, 15, 16). However, when considering the potential to mutate to 1 of the 6 non-synonymous key contact residues (Arg, Asn, Asp, Gly, Ser, Thr, and Tyr), AGY Ser codons are able to do so via 12 out of 18 possible single-base changes. For AAY (Asn), this occurs with 8 out of 18 base changes, and for TCN, it occurs with only 6 out of 36 base substitutions (Figure 5), a result that is in agreement with the observation by Chang and Casali that CDR, but not FR sequences, are prone to acquire replacement mutations upon random point mutation (41). Collectively, the results of these analyses indicate that AGY codons contribute to Ab affinity both directly, by encoding a Ser residue, and indirectly due to the ease with which they mutate to encode other residues beneficial to the process of Ab affinity maturation. We believe this is the most straightforward explanation for the conservation of AGY codon abundance in CDRs of germline IgV-region genes.\nFigure 5 AGY Ser codons plasticity. Probability of creating a key non-synonymous contact residue by one nucleotide change. Filled gray boxes indicate a key Ag-contact residue as defined by Raghunathan et al. (19). White boxes indicate a synonymous change, a non-key contact residue (defined in the text) or a stop codon."}