A comparative evolutionary approach to enzyme characterization: AID and AID-like enzymes in early-evolved species

Quinlan, Emma M. (2022) A comparative evolutionary approach to enzyme characterization: AID and AID-like enzymes in early-evolved species. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The immunoglobulin (Ig)-based adaptive immune system (AIS) evolved ~ 500 million years ago in jawed vertebrates, while the jawless vertebrates exhibit their own variable lymphocyte receptor (VLR)-based AIS. An evolutionarily conserved hallmark of the Ig-based AIS is the diversification of Ig-class antibodies through somatic hypermutation and/or class switch recombination of Ig genes, initiated by the DNA-mutating enzyme activation-induced cytidine deaminase (AID). The VLR-based AIS may also involve activities of AID-like enzymes, but their specific activities have remained unclear. Each clade of vertebrate fishes possesses genetically diverse AID and AID-like enzymes, whose biochemical characteristics, and in some cases functionality, were not known. To elucidate structure-function relationships and to biochemically characterize these distinct proteins, I examined AID homologs from the sea and freshwater lampreys, nurse shark, zebrafish, tetraodon, coelacanth, and human. First, I discovered that the nurse shark and coelacanth, both species representative of significant evolutionary junctures of vertebrate speciation, posses an active AID enzyme. Second, I found the highly unusual human AID biochemical properties of lethargic activity and high affinity binding to its single-stranded DNA substrate were conserved across all vertebrates, while other characteristics, like optimal temperature and sequence preference, diverged. Interestingly, while most vertebrates have only one AID gene, the freshwater lampreys were found to have multiple AID-like genes, grouped into cytidine deaminase 1 (CDA1), CDA1-like, and CDA2 genes, based on the CDA1 and CDA2 genes found previously in the sea lamprey. Furthermore, each individual expresses different combinations of the AID-like genes. The CDA1 and CDA1-like enzymes exhibited varying pH sensitivities and enzyme activity levels, similar to the differences between human AID and its APOBEC family member enzymes, thus suggesting unique roles for the CDA1 and CDA1-like enzymes in the lamprey AIS. Faint activity was detected for the sea lamprey CDA2, similar to ongoing, yet unpublished, efforts by other research groups. This is the first study to biochemically and structurally characterize AID and AID/APOBEC-like enzymes covering the span of fish evolution. This multidimensional approach of multi-species in silico structural analysis and in vitro biochemical characterization exemplifies a comprehensive method for gaining deep insight into enzyme activity, function, and evolution.

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