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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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Changing the target specificity of AID. (A) Depiction of AID cDNA showing the Zn coordination domain (HVE and PCYDC), and the region containing a putative substrate contact loop (gray), with alignment to equivalent regions of APOBEC3C, 3F, and 3G shown below. Residues predicted to be in the substrate contact loop are highlighted in bold. In the AID chimeras, residues 115–123 of AID were replaced by equivalent residues in APOBEC3C/F/G as indicated. (B) Bacterial mutator activity of variant AIDs was determined by the mean frequency from 12 independent cultures with which they yielded colonies resistant to rifampicin (Rifr), expressed relative to that given by the vector-only control. AID1 and AID2 are previously described upmutants of wild-type AID (AID1: K10E/T82I/E156G; AID2: K34E/E156G/R157T; Wang et al., 2009). (C) Target specificity of the various AID-derived deaminases as judged by the distribution of rpoB mutations in rifr resistant colonies. Transition mutations at any one of eleven C:G pairs within rpoB can give rise to Rifr. Mutations at a specific C:G pair are expressed as a percentage of the total number of Rifr colonies scored for each deaminase. Results with AID/3G are shaded black, AID1/3G in dark gray, and AID2/3G in light gray (all largely target C1691).
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fig1: Changing the target specificity of AID. (A) Depiction of AID cDNA showing the Zn coordination domain (HVE and PCYDC), and the region containing a putative substrate contact loop (gray), with alignment to equivalent regions of APOBEC3C, 3F, and 3G shown below. Residues predicted to be in the substrate contact loop are highlighted in bold. In the AID chimeras, residues 115–123 of AID were replaced by equivalent residues in APOBEC3C/F/G as indicated. (B) Bacterial mutator activity of variant AIDs was determined by the mean frequency from 12 independent cultures with which they yielded colonies resistant to rifampicin (Rifr), expressed relative to that given by the vector-only control. AID1 and AID2 are previously described upmutants of wild-type AID (AID1: K10E/T82I/E156G; AID2: K34E/E156G/R157T; Wang et al., 2009). (C) Target specificity of the various AID-derived deaminases as judged by the distribution of rpoB mutations in rifr resistant colonies. Transition mutations at any one of eleven C:G pairs within rpoB can give rise to Rifr. Mutations at a specific C:G pair are expressed as a percentage of the total number of Rifr colonies scored for each deaminase. Results with AID/3G are shaded black, AID1/3G in dark gray, and AID2/3G in light gray (all largely target C1691).

Mentions: The human APOBEC3 proteins, like AID, are able to deaminate C in DNA, but whereas AID prefers to target C residues flanked by a 5′-flanking purine, the APOBEC3s largely prefer a 5′-pyrimidine flank with individual APOBEC3s differing with regard to the details of this 5′-flanking nucleotide preference. Comparison of human APOBEC3 gene sequences previously led us to speculate that a stretch of ∼8 amino acids located some 60 residues from the C-terminal end of the protein domain played an important role in determining this flanking nucleotide preference; we supported this speculation by showing that the target specificity of APOBEC3F could indeed be altered by specific amino acid changes in this region (Langlois et al., 2005). Subsequently, in light of the crystal structure of APOBEC2 (Prochnow et al., 2007) together with that of the TadA tRNA-adenosine deaminase in complex with an oligonucleotide substrate (Losey et al., 2006), we suggested this amino acid stretch in both AID and APOBEC3s likely formed a contact with the DNA substrate (Conticello et al., 2007). Thus, to generate AID variants with altered target specificity, we focused on this stretch of amino acids and replaced AID residues 115–123 with the corresponding portion of APOBEC3s (Fig. 1 A). Kohli et al. (2009) have recently adopted a similar approach to modify AID’s substrate preference.


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)

Changing the target specificity of AID. (A) Depiction of AID cDNA showing the Zn coordination domain (HVE and PCYDC), and the region containing a putative substrate contact loop (gray), with alignment to equivalent regions of APOBEC3C, 3F, and 3G shown below. Residues predicted to be in the substrate contact loop are highlighted in bold. In the AID chimeras, residues 115–123 of AID were replaced by equivalent residues in APOBEC3C/F/G as indicated. (B) Bacterial mutator activity of variant AIDs was determined by the mean frequency from 12 independent cultures with which they yielded colonies resistant to rifampicin (Rifr), expressed relative to that given by the vector-only control. AID1 and AID2 are previously described upmutants of wild-type AID (AID1: K10E/T82I/E156G; AID2: K34E/E156G/R157T; Wang et al., 2009). (C) Target specificity of the various AID-derived deaminases as judged by the distribution of rpoB mutations in rifr resistant colonies. Transition mutations at any one of eleven C:G pairs within rpoB can give rise to Rifr. Mutations at a specific C:G pair are expressed as a percentage of the total number of Rifr colonies scored for each deaminase. Results with AID/3G are shaded black, AID1/3G in dark gray, and AID2/3G in light gray (all largely target C1691).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2812546&req=5

fig1: Changing the target specificity of AID. (A) Depiction of AID cDNA showing the Zn coordination domain (HVE and PCYDC), and the region containing a putative substrate contact loop (gray), with alignment to equivalent regions of APOBEC3C, 3F, and 3G shown below. Residues predicted to be in the substrate contact loop are highlighted in bold. In the AID chimeras, residues 115–123 of AID were replaced by equivalent residues in APOBEC3C/F/G as indicated. (B) Bacterial mutator activity of variant AIDs was determined by the mean frequency from 12 independent cultures with which they yielded colonies resistant to rifampicin (Rifr), expressed relative to that given by the vector-only control. AID1 and AID2 are previously described upmutants of wild-type AID (AID1: K10E/T82I/E156G; AID2: K34E/E156G/R157T; Wang et al., 2009). (C) Target specificity of the various AID-derived deaminases as judged by the distribution of rpoB mutations in rifr resistant colonies. Transition mutations at any one of eleven C:G pairs within rpoB can give rise to Rifr. Mutations at a specific C:G pair are expressed as a percentage of the total number of Rifr colonies scored for each deaminase. Results with AID/3G are shaded black, AID1/3G in dark gray, and AID2/3G in light gray (all largely target C1691).
Mentions: The human APOBEC3 proteins, like AID, are able to deaminate C in DNA, but whereas AID prefers to target C residues flanked by a 5′-flanking purine, the APOBEC3s largely prefer a 5′-pyrimidine flank with individual APOBEC3s differing with regard to the details of this 5′-flanking nucleotide preference. Comparison of human APOBEC3 gene sequences previously led us to speculate that a stretch of ∼8 amino acids located some 60 residues from the C-terminal end of the protein domain played an important role in determining this flanking nucleotide preference; we supported this speculation by showing that the target specificity of APOBEC3F could indeed be altered by specific amino acid changes in this region (Langlois et al., 2005). Subsequently, in light of the crystal structure of APOBEC2 (Prochnow et al., 2007) together with that of the TadA tRNA-adenosine deaminase in complex with an oligonucleotide substrate (Losey et al., 2006), we suggested this amino acid stretch in both AID and APOBEC3s likely formed a contact with the DNA substrate (Conticello et al., 2007). Thus, to generate AID variants with altered target specificity, we focused on this stretch of amino acids and replaced AID residues 115–123 with the corresponding portion of APOBEC3s (Fig. 1 A). Kohli et al. (2009) have recently adopted a similar approach to modify AID’s substrate preference.

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