Abstract
The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing AID-deficient primary mouse B cells. The combination of the three methods, accompanied by the necessary analysis of AID subcellular localization and protein expression levels and stability, as controls, allows detailed structure-function interrogation of AID.
Keywords: Activation induced deaminase, Mutation, Class switch recombination, Escherichia coli, B-lymphocytes
Part I. Measuring mutagenic activity of AID in E. coli The following procedure has been adapted from Petersen-Mahrt et al. (2002). It provides a measurement of the capacity of AID to mutate the Escherichia coli genome by selecting for those mutations in the rpoB gene that confer resistance to Rifampicin. It can be used as a proxy for the catalytic activity of the enzyme, although it involves deamination of a transcribed gene, which might influence the results. When comparing different AID variants or mutants, it provides a measurement of their mutagenic activity compared to the wt enzyme, which often but not always correlates with the enzymatic activity of the corresponding recombinant enzymes measured on DNA oligonucleotide substrates.
Materials and Reagents
Equipment
Procedure
Prepare as many 10 cm LB agar plates supplemented with rifampicin (100 μg/ml) + ampicillin (100 μg/ml) as samples will be tested (selection plates). Prepare also the necessary number of LB agar plates supplemented with ampicillin (100 μg/ml) for the initial transformation and for viability plates in the experiment (see below). For example, to compare 3 constructs prepare 3 plates supplemented with ampicillin for bacterial transformation plus 3 x 5 = 15 plates supplemented with ampicillin for the assay (for a total of 18 ampicillin plates), as well as 15 plates supplemented with rifampicin + ampicillin.
Recipes
Note: Media must be autoclaved after preparation and kept sterile.
Part II. Measuring somatic hypermutation activity of AID in DT40 cells DT40 cells are a chicken B cell lymphoma line that constitutively expresses IgM at the plasma membrane, as well as AID (Arakawa et al., 2002). Chickens diversify their Ig genes by gene conversion, a homologous recombination-based mechanism. However, the DT40-derived cell line used here (DT40 ΔΨV IgM+) has been engineered to eliminate the V pseudogenes that act as homology donors for gene conversion, causing that AID deaminations accumulate as mutations, mostly at C:G pairs with a bias towards transversion mutations (Arakawa et al., 2004). The protocol below takes advantage of the fact that a fraction of AID-induced mutations at the Ig loci will disrupt IgM expression. This can be quantified by flow cytometry as IgM-loss cells, and is a convenient proxy to monitor SHM frequency. When starting from a DT40 ΔΨV IgM+ population, the proportion of IgM-loss cells is a function of the time the cells spent in culture as well as of the SHM frequency, which depends directly on AID activity. A DT40 ΔΨV IgM+ line in which AID expression has also been eliminated by knock out (KO) can be complemented with expression vectors encoding AID or AID mutants, either tagged with GFP or linked to GFP expression by an internal ribosomal entry site, in order to compare their relative ability for SHM. GFP expression is used to monitor infection efficiency and to evaluate lgM-loss on infected cells only.
Part III. Measuring AID class switch recombination activity This method uses retroviral complementation of primary B cells obtained from the spleen of AID-deficient (Aicda-/-) mice, to assay the capacity of AID variants to produce CSR.
Note: All procedures are performed in a tissue culture hood to prevent contaminations.
Acknowledgments
The protocols described here were adapted from our publications (Methot et al., 2015; Zahn et al., 2014). Our studies are supported in part by the Canadian Institutes of Health Research, the Cancer Research Society Inc and a Canada Research Chair tier 2 to JMDN. LCL is supported in part by a Cole Foundation Fellowship and SPM is supported by a fellowship from Fonds de recherche Quebec-Santé (FRQ-S).
References
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