"
Team:Potsdam Bioware/Project/Part AID
From 2012.igem.org
Part AID
Activation-induced cytidine deaminase (AID)
Overview
Introduction
The activation-induced cytidine deaminase (AID) is one of the key enzymes of mammalian immune system and a central controlling point for the somatic hypermutation (SHM), class switch recombination and gene conversion. The AID is expressed in B-lymphocytes and active during B-lymphocyte activation. Because of sequence similarities to APOBEC (apolipoprotein B messenger RNA-editing enzyme catalytic polypetide) enzymes many functional and structural aspects of the AID were postulated. Today we know that the AID catalysis the deamination of cytidine to uracil on the DNA level. A deficiency of AID or mutations within the coding sequence causes a defect of antibody maturation, class switch recombination and gene conversion.
Structure of the AID
The AID is a 28 kDa small protein with a highly conserved structure. The three-dimensional structure was not clear up until today. But because of structural homologies to the APOBEC enzymes some structural aspects were suggested. However, it is known that the AID has a non-functional N-terminal nuclear location sequence (NLS) and a C-terminal nuclear export sequence (NES). In the middle of the primary sequence the cytidine motif and the APOBEC-like motif is localized. Because of simulations it was suggested that the AID is a dimer and every subunit binds to the DNA.
Substrate specificity of the AID
It was shown that the AID mutates DNA instead of RNA in case of APOBEC enzymes. The substrate of the AID is the singlestranded DNA. Therefore a correlation between the mutation rate and the transcription rate existed which means that the greater the transcription activity the higher the mutation rate. That’s the reason why the AID has to be localized in the nucleus of eukaryotic cells to mutate the DNA sequence. Because of the NES the enzyme is actively exported. Nevertheless the small size of the AID allows it to diffuse through the nuclear pore complexes into the nucleus.
The SHM should take place on the variable regions (V-regions) of the antibodies; the binding sites to the antigen. Therefore the AID should bind specific to the V-regions and catalysis the mutation. However, the SHM was also observed in the genes bcl-6 and FasL albeit with a much lower mutation rate. Consequnetly, it was suggested that the AID has no direct substrate specificity and maybe interact with some cis acting factors to ensure a significantly greater mutation rate in the V-regions than in other genes. After sequence analysis of the mutated target sequences is was found out that the AID mutates predominantly small sequence motifs; so called hotspots.
AID as a key component of the Antibody generating system
In a prior publication it was shown that the AID can be used to maturate antibody sequences to produce high affine binding molecules in CHO cells. In our project we also use the AID to mutate the antibody sequences in CHO cells. Therefore we cloned the AID biobrick (BBa_K103001) with the CMV promoter and the hGH-polyA tail together to transfect transiently the CHO cells. We expected that the mutation rate is depending on the predominantly location of this enzyme in the cell. Therefore we concluded that the mutation rate increase with the increasing resting time of the AID in the nucleus. Therefore we knocked out the C-terminal NES and add a functional NLS to the N-terminus to ensure a greater resting time in the nucleus. To investigate whether the modifications are functional we fused the modified AID with the eGFP.
For the Phage-Display we use the AID cloned in the pBAD-vector backbone to transform Escherichia coli cells. In this case the transcription of the AID was influenced by the arabinose promoter to avoid a leaky expression of this enzyme.
