Analysis of diversity of nucleotide and amino acid distributions in the VD and DJ joining regions in Ig heavy chains.
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Nucleotide fill-in between the germ line V, D and J genes in the H3 loop of immunoglobulins contributes to the diversity of the antibody repertoire. This fill-in process is mediated by terminal deoxynucleotidyl transferase (TdT), which has been widely believed to insert nucleotides in a random fashion. Using a database of 2443 immunoglobulin sequences, we identified the regions of nucleotide fill-in between the V-D and D-J gene regions. We translated the fill-in nucleotides and measured the diversity within the two regions both at the nucleotide and amino acid level. We found that the nucleotide and amino acid distributions that resulted from nucleotide fill-in were in fact not random. Examination of the synonymous substitution rates of nucleotides revealed evidence suggesting that TdT plays a less significant role in generating antibody diversity than previously thought. We observed preferences for polar residues, which are more likely to encourage interaction with ligand than non-polar residues and are often found in loop regions in general. We also observed a preference for the insertion of smaller residues versus larger residues of similar biochemical properties, aiding in loop flexibility. We interpret these findings to reflect the significant influence of biochemical (i.e. folding) constraints and/or binding affinity constraints (at the cellular/selectional level) on the sequence diversity in the H3 region. These constraints act as a filter on the randomness generated by nucleotide addition by TdT, as well as other diversity generating processes such as recombination of VDJ gene segments and somatic mutation. The results of this study suggest that the antibody repertoire might be reduced from what is traditionally believed.