Activation induced cytidine deaminase mutant (AID-His130Pro) from Hyper IgM 2 patient retained mutagenic activity on SHM artificial substrate

Hanen Ouadani,Dahmani Fathallah,Tihana Jovanic,Sylvie Garcia,François Rougeon,Mongia Hachicha,Benoit Arcangioli,Mohamed-Ridha Barbouche,Imen Ben-Mustapha,Houda Elloumi-Zghal,Meriem Ben-Ali,Hatem Masmoudi,Beya Larguèche

doi:10.1016/j.molimm.2016.09.025

Abstract

Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) during secondary immune response. Mutations in the AICDA gene are responsible for Hyper IgM 2 syndrome where both CSR and SHM or only CSR are affected. Indeed, triggering either of the two mechanisms requires the DNA deamination activity of AID. Besides, different domains of AID may be differentially involved in CSR and SHM through their interaction with specific cofactors. Herein, we studied the AID-induced SHM activity of the AID-His130Pro mutant identified in a patient with Hyper IgM 2 syndrome. AID mutagenic activity was monitored by the reversion of nonsense mutations of the EGFP gene assessed by flow cytometry. We found that the His130Pro mutation, which affects CSR, preserves AID mutagenic activity. Indeed, the His130 residue is located in a putative specific CSR region in the APOBEC-like domain, known to involve CSR specific cofactors that probably play a major role in AID physiological activities.

Highlights

Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) are the mechanisms that allow antibodies to ensure diverse effector functions and to reach affinity maturation
The His130Pro nonsynonymous mutation was predicted to be benign by both PolyPhen2 and SIFT algorithms suggesting that the His130Pro mutation does not affect drastically the protein structure
Western blot analysis of EBV lymphoblastoide cell lines (LCLs) lysates from Hyper IgM 2 (HIGM2) patient, carrying the His130Pro mutation, revealed a low expression level of the mutant Activation induced cytidine deaminase (AID)-His130Pro protein compared to the wild type

Summary

Introduction

Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) are the mechanisms that allow antibodies to ensure diverse effector functions and to reach affinity maturation. SHM results mainly from single nucleotide substitutions within the variable (V) genes of immunoglobulins (Peled et al, 2008) These two mechanisms are initiated by the enzyme Activation Induced Cytidine Deaminase (AID) following interaction between CD40 and CD40L expressed on B cells and activated T cells respectively (Crouch et al, 2007; Muramatsu et al, 2000). AID shares an APOBEC-like domain with the cytidine deaminase family. Analyses of AID mutant proteins activity in HIGM2 patients allow a better understanding of CSR and SHM targeting mechanism. AID C-terminal mutations display a defect in CSR with normal SHM activity showing that this domain is CSR specific (Durandy et al, 2006). We aim to assess the SHM activity of AID-His130Pro mutant using an in vitro mutagenesis assay on an artificial SHM substrate already developed by Rougeon and collaborators (Jovanic et al, 2008)

Methods

Results

Conclusion