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. 2012;7(2):e30667.
doi: 10.1371/journal.pone.0030667. Epub 2012 Feb 20.

Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

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Free PMC article

Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

Roland Geisberger et al. PLoS One. .
Free PMC article

Abstract

Background: Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive.

Methodology/principal findings: Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells.

Conclusions/significance: Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of lysine to arginine mutations in the AID protein on CSR activity.
(A) Schematic representation of AID highlighting the position of the lysine (K) residues. For CSR assays, untagged constructs were expressed bicistronically together with GFP separated by an internal ribosome entry site (IRES). (B) Representative FACS profile for CSR activity of individual Lys to Arg mutants. B cells from AID deficient mice were retrovirally transfected with the respective lysine AID mutant (Mx: empty vector) and CSR to IgG1 was determined upon stimulation of cells with LPS and IL-4. GFP expression of infected cells was driven from an IRES element located 3′ of the stop codon of the cloned AID mutant. The percentage in the upper right quadrants indicate the percentage of IgG1+ cells among the GFP+ (i.e. retrovirally infected) population. (C) Results from three independent experiments of a screen of lysine mutants showing the percentage of IgG1+ cells among the GFP+ population. (D) Results from 11 independent experiments comparing the CSR activity of AID and K3. (E) Western Blot on lysates from B cells retrovirally transfected with the respective constructs. Black arrowheads indicate specific band. White arrowheads indicate nonspecific band. (IB: immune blot).
Figure 2
Figure 2. Mutational activity of Lys22 mutants.
Rifampicin (Rif) resistance assay for AID-mediated cytidine deamination in UNG-deficient BW310 E.coli. The graph shows the mutation frequency (defined as the frequency of Rif-resistant clones) of individual starting colonies from E.coli transfected with the empty vector or plasmids expressing wt AID, K3 or lysineless deltaK. Median values ± SD are indicated in the graph.
Figure 3
Figure 3. Subcellular localization of K3 is not different from wildtype AID.
(A) Schematic representation of AID-GFP fusion constructs used for localization assays. GFP is fused to the C-terminus of AID or mutant K3. (B) HEK293T cells were transiently transfected with the respective GFP-fusion constructs. Two days post transfection, cells were treated as indicated with Leptomycin B (LMB) for 3 hours. Cells were fixed and localization of the respective GFP fusion proteins visualized using fluorescence microscopy. (C) The k46 B cell line stably expressing AID-GFP or K3-GFP fusion proteins were treated with or without Leptomycin B (LMB) for 3 hours and localization of GFP fusion constructs was determined using fluorescence microscopy.
Figure 4
Figure 4. Somatic Hypermutation of IgV genes by wt AID and mutant K3.
(A) Somatic hypermutation reflected as loss of IgM expression in AID−/− ΨV−/− IgM+ DT40 cells. AID−/− ΨV−/− IgM+ DT40 cells were stably transfected with plasmids encoding AID or K3 coupled to IRES-GFP expression. The percentage of sIgM-loss variants of GFP+ cells is expressed as the median ± SE of multiple (n) independent clonal transfectants determined three weeks after transfection (untransfected control, n = 12; AID, n = 36; K3, n = 44). (B) Proportion of sequences carrying mutations in IgV genes of DT40 cells. IgV regions were cloned from cDNA of AID and K3 expressing DT40 transfectants. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of each chart. The frequency of mutations per bp sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart, respectively. (C) The abundance of AID and K3 in DT40 transfectants was determined by Western blotting from cell extracts. Blots were reprobed for GFP expression and actin levels as loading control. (D) Proportion of sequences carrying Bcl6 mutations in AID deficient B cells retrovirally transfected with AID or K3 constructs, respectively. Bcl6 was cloned from genomic DNA of sorted mouse B cells, retrovirally transfected to express AID or K3, respectively. Pie charts were generated as described in (B).

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