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Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

Wang M, Rada C, Neuberger MS - J. Exp. Med. (2010)

Bottom Line: The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide.As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine.Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, England, UK.

ABSTRACT
High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

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Modified AIDs give altered IgVλ hypermutation spectra in B cells. (A) Hypermutation of IgVλ was assayed by monitoring surface IgM-loss in AID−/− ψV−/− sIgM+ DT40 cells that had been stably transfected with constructs coexpressing the indicated AID mutants together with GFP. For each construct, the percentage of surface IgM-loss variants in 8–12 independent clonal transfectants were determined 3 wk after subculturing. On the right, Western blots representative of multiple clones show AID abundance in the DT40 cell extracts, with tubulin as loading control. (B) 5′-flanking nucleotide preferences of the IgVλ C mutations produced by the variant AID deaminases in the DT40 clonal transfectants. The compilations are based on mutations detected in unsorted DT40 cells analyzed 8 wk after transfection, except for AID/3C, where sequences from both unsorted and sorted sIgM− populations contributed to the mutation database. In the case of AID1*/3G, the nucleotide preferences are given based on an analysis of all the mutations in the dataset, as well as from an analysis in which the four major hotspots were removed from the calculations. (C) Percentage of mutated C residues flanked by 5′-purine (red) or 5′-pyrimidine (blue) in IgVλ sequences analyzed from individual expanded DT40 clonal transfectants represented by each bar. (D) Distribution of IgVλ mutations in the DT40 transfectants, in each comparing the spectrum achieved with a modified AID (below the line) to that achieved with wild-type AID (above the line). Mutations (which were >95% at C:G pairs) were computed as being caused by C deamination with those Cs flanked by a 5′-purine (Pu-C) indicated in red and those by a 5′-pyrimidine (Py-C) in blue. Further details on the mutations obtained with these deaminases, as well as with AID1 are shown in Fig. S2 and Fig. S3.
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fig3: Modified AIDs give altered IgVλ hypermutation spectra in B cells. (A) Hypermutation of IgVλ was assayed by monitoring surface IgM-loss in AID−/− ψV−/− sIgM+ DT40 cells that had been stably transfected with constructs coexpressing the indicated AID mutants together with GFP. For each construct, the percentage of surface IgM-loss variants in 8–12 independent clonal transfectants were determined 3 wk after subculturing. On the right, Western blots representative of multiple clones show AID abundance in the DT40 cell extracts, with tubulin as loading control. (B) 5′-flanking nucleotide preferences of the IgVλ C mutations produced by the variant AID deaminases in the DT40 clonal transfectants. The compilations are based on mutations detected in unsorted DT40 cells analyzed 8 wk after transfection, except for AID/3C, where sequences from both unsorted and sorted sIgM− populations contributed to the mutation database. In the case of AID1*/3G, the nucleotide preferences are given based on an analysis of all the mutations in the dataset, as well as from an analysis in which the four major hotspots were removed from the calculations. (C) Percentage of mutated C residues flanked by 5′-purine (red) or 5′-pyrimidine (blue) in IgVλ sequences analyzed from individual expanded DT40 clonal transfectants represented by each bar. (D) Distribution of IgVλ mutations in the DT40 transfectants, in each comparing the spectrum achieved with a modified AID (below the line) to that achieved with wild-type AID (above the line). Mutations (which were >95% at C:G pairs) were computed as being caused by C deamination with those Cs flanked by a 5′-purine (Pu-C) indicated in red and those by a 5′-pyrimidine (Py-C) in blue. Further details on the mutations obtained with these deaminases, as well as with AID1 are shown in Fig. S2 and Fig. S3.

Mentions: Mutation spectrum of modified AIDs assayed in vitro on a gapped duplex lacZ target. (A) Depiction of the M13mp19lacZ gapped duplex substrate DNA. (B) 5′-flanking nucleotide preferences of the C mutations produced by the variant AID deaminases. The spectra shown for the AID/3G chimera is from an AID1*/3G derivative in which the asterisk denotes that the protein has been truncated at amino acid position 190 of AID (removing the nuclear export sequence) and is shown to allow comparison with the same AID variant analyzed in transfected DT40 B cells (see Fig. 3). The C-terminal truncations do not detectably alter the patterns of in vitro mutational targeting. (C) Distribution of mutations over a 310-nt stretch of the single-stranded lacZ target. The numbers of independent mutations at each nucleotide position are expressed as a percentage of the total mutation database (as analyzed over the entire 475-nt single-stranded target). Nucleotide position 1 is defined as the start of the lac promoter. Mutations at C residues flanked by a 5′-purine (Pu-C) are shown in red, those flanked by a 5′-pyrimidine (Py-C) in blue. (D) Identity of the three most frequently targeted residues by each deaminase, with targeting expressed as the percentage of clones analyzed in which the relevant cytosine (underlined) was mutated.


Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

Wang M, Rada C, Neuberger MS - J. Exp. Med. (2010)

Modified AIDs give altered IgVλ hypermutation spectra in B cells. (A) Hypermutation of IgVλ was assayed by monitoring surface IgM-loss in AID−/− ψV−/− sIgM+ DT40 cells that had been stably transfected with constructs coexpressing the indicated AID mutants together with GFP. For each construct, the percentage of surface IgM-loss variants in 8–12 independent clonal transfectants were determined 3 wk after subculturing. On the right, Western blots representative of multiple clones show AID abundance in the DT40 cell extracts, with tubulin as loading control. (B) 5′-flanking nucleotide preferences of the IgVλ C mutations produced by the variant AID deaminases in the DT40 clonal transfectants. The compilations are based on mutations detected in unsorted DT40 cells analyzed 8 wk after transfection, except for AID/3C, where sequences from both unsorted and sorted sIgM− populations contributed to the mutation database. In the case of AID1*/3G, the nucleotide preferences are given based on an analysis of all the mutations in the dataset, as well as from an analysis in which the four major hotspots were removed from the calculations. (C) Percentage of mutated C residues flanked by 5′-purine (red) or 5′-pyrimidine (blue) in IgVλ sequences analyzed from individual expanded DT40 clonal transfectants represented by each bar. (D) Distribution of IgVλ mutations in the DT40 transfectants, in each comparing the spectrum achieved with a modified AID (below the line) to that achieved with wild-type AID (above the line). Mutations (which were >95% at C:G pairs) were computed as being caused by C deamination with those Cs flanked by a 5′-purine (Pu-C) indicated in red and those by a 5′-pyrimidine (Py-C) in blue. Further details on the mutations obtained with these deaminases, as well as with AID1 are shown in Fig. S2 and Fig. S3.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812546&req=5

fig3: Modified AIDs give altered IgVλ hypermutation spectra in B cells. (A) Hypermutation of IgVλ was assayed by monitoring surface IgM-loss in AID−/− ψV−/− sIgM+ DT40 cells that had been stably transfected with constructs coexpressing the indicated AID mutants together with GFP. For each construct, the percentage of surface IgM-loss variants in 8–12 independent clonal transfectants were determined 3 wk after subculturing. On the right, Western blots representative of multiple clones show AID abundance in the DT40 cell extracts, with tubulin as loading control. (B) 5′-flanking nucleotide preferences of the IgVλ C mutations produced by the variant AID deaminases in the DT40 clonal transfectants. The compilations are based on mutations detected in unsorted DT40 cells analyzed 8 wk after transfection, except for AID/3C, where sequences from both unsorted and sorted sIgM− populations contributed to the mutation database. In the case of AID1*/3G, the nucleotide preferences are given based on an analysis of all the mutations in the dataset, as well as from an analysis in which the four major hotspots were removed from the calculations. (C) Percentage of mutated C residues flanked by 5′-purine (red) or 5′-pyrimidine (blue) in IgVλ sequences analyzed from individual expanded DT40 clonal transfectants represented by each bar. (D) Distribution of IgVλ mutations in the DT40 transfectants, in each comparing the spectrum achieved with a modified AID (below the line) to that achieved with wild-type AID (above the line). Mutations (which were >95% at C:G pairs) were computed as being caused by C deamination with those Cs flanked by a 5′-purine (Pu-C) indicated in red and those by a 5′-pyrimidine (Py-C) in blue. Further details on the mutations obtained with these deaminases, as well as with AID1 are shown in Fig. S2 and Fig. S3.
Mentions: Mutation spectrum of modified AIDs assayed in vitro on a gapped duplex lacZ target. (A) Depiction of the M13mp19lacZ gapped duplex substrate DNA. (B) 5′-flanking nucleotide preferences of the C mutations produced by the variant AID deaminases. The spectra shown for the AID/3G chimera is from an AID1*/3G derivative in which the asterisk denotes that the protein has been truncated at amino acid position 190 of AID (removing the nuclear export sequence) and is shown to allow comparison with the same AID variant analyzed in transfected DT40 B cells (see Fig. 3). The C-terminal truncations do not detectably alter the patterns of in vitro mutational targeting. (C) Distribution of mutations over a 310-nt stretch of the single-stranded lacZ target. The numbers of independent mutations at each nucleotide position are expressed as a percentage of the total mutation database (as analyzed over the entire 475-nt single-stranded target). Nucleotide position 1 is defined as the start of the lac promoter. Mutations at C residues flanked by a 5′-purine (Pu-C) are shown in red, those flanked by a 5′-pyrimidine (Py-C) in blue. (D) Identity of the three most frequently targeted residues by each deaminase, with targeting expressed as the percentage of clones analyzed in which the relevant cytosine (underlined) was mutated.

Bottom Line: The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide.As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine.Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, England, UK.

ABSTRACT
High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Show MeSH
Related in: MedlinePlus