Evolutionary trajectory of the enzyme activation-induced cytidine deaminase (AID) within the Gadiformes lineage

Ghorbani, Atefeh (2021) Evolutionary trajectory of the enzyme activation-induced cytidine deaminase (AID) within the Gadiformes lineage. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Activation-induced cytidine deaminase (AID) is a DNA-mutating enzyme that initiates secondary antibody diversification process upon immune stimulation. One outcome of this diversification is the generation of antibodies with higher affinity for the cognate antigen. In human and mouse models, functional deficiency of AID leads to hyper IgM syndrome type Ⅱ, exhibiting lack of secondary antibody diversification. Despite the central role of AID in instigating this diversification process, its off-targeting activity has been attributed to the initiation and progression of various type of cancers. The emergence of AID and, therefore, secondary antibody diversification process have been dated back to the common ancestor of jawed vertebrates. However, several studies investigating the Atlantic cod (Gadus morhua) immune responses revealed lack of high affinity antibodies and robust humoral response in this species. Moreover, genomic sequence of several Gadiformes species, including Atlantic cod, uncovered the loss of histocompatibility complex class II (mhc Ⅱ), cluster of differentiation 4 (cd4), and invariant chain (Ii) genes in their common ancestor. These genes are involved in B cell activation in the mammalian model of immune system. Since AID is responsible for generation of high affinity antibodies in other vertebrates, we sought to examine the genetics, expression, and function of Atlantic cod AID. We also investigated the evolutionary trajectory of AID within Gadiformes species to shed light on the extent of immune system remodeling in this lineage. In chapter two, we showed that the AID gene synteny and transcript expression were conserved in Atlantic cod in comparison with other studied vertebrates. Interestingly, we identified two distinct AID transcripts, one of which encoded a full-length AID, whilst the other one lacked the first exon. In chapter three, we synthesized, expressed, and purified Atlantic cod AID (Gm-AID) and examined its biochemical properties. Our results showed that despite having a similar DNA binding ability, Gm-AID exhibited extremely low catalytic efficiency compared with other studied vertebrates. In chapter four, we synthesized, expressed, and purified 36 AID homologs within and outside of the Gadiformes lineage. Previous studies have shown a drastic re-modeling of the Gadigormes’ immune system where the loss of genes involved in antibody responses has coincided with an expansion of innate and cell-mediated immune genes. Our biochemical analyses revealed a vast diversity in the enzymatic properties of AID homologs. Remarkably, two Gadifomes AID homologs examined here did not exhibit any cytidine deaminase activity. By predicting and resurrecting the ancestral AIDs within and outside of Gadiformes lineage, we showed that the functional impairment of AID most likely has happened in the ancestor of Gadidae group. Since Gadidae species have successfully populated their natural habitats, the functional impairment of their AID enzyme did not hamper their fitness. This is most likely duet to the compensatory mechanisms such as the expansion of innate and cell-mediated immune systems. Our findings of the first example of a vertebrate species with a dysfunctional AID and secondary antibody diversification challenge the long-standing immunological concept that the loss of AID activity leads to immunodeficiency.

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