Abstract
AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C --> U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.
Highlights
JULY 30, 2010 VOLUME 285 NUMBER 31 initiated somatic hypermutation (SHM) within immunoglobulin variable (IgV) regions and concomitant class switch recombination in Ig switch regions (S regions) [3]
The association of AID and GFP-GANP was not affected by treatment with RNase A or DNase I (Fig. 1C), suggesting that AID and GANP associate through protein-protein interactions
These results show that AID and GANP form a complex in COS-7 cell nuclei, implying the likelihood that a complex containing GANP and AID may occur in activated B-cell nuclei
Summary
Construction of Mammalian Expression Vectors—Mammalian expression vectors were constructed as follows. COS-7 cells (1 ϫ 106 cells) were mixed with each expression vector (1.5 g for GFP alone and DsRed fusion proteins, 4.5 g for GFPGANP) and transfected by using Amaxa’s Nucleofection kit RTM (program O-01) according to the manufacturer’s protocols. For in vitro binding assay, each 2 l of WGE was mixed in TNE buffer and precipitated with anti-FLAG Ab. Mutant AID Purification and Activity Assay—Recombinant wild type and mutant AID (D143A) proteins were expressed in baculovirus-infected Sf9 cells and purified as described previously [6, 7]. Washed cells were lysed in radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS) supplemented with protease inhibitors and were sonicated with three rounds of pulses with 30-s intervals with a BioruptorTM (Cosmo Bio Corp.). A value of p Ͻ 0.05 was considered to be of statistical significance
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