Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish

Abstract

Background Antibody affinity maturation in vertebrates requires the enzyme activation-induced cytidine deaminase (AID) which initiates secondary antibody diversification by mutating the immunoglobulin loci. AID-driven antibody diversification is conserved across jawed vertebrates since bony and cartilaginous fish. Two exceptions have recently been reported, the Pipefish and Anglerfish, in which the AID-encoding aicda gene has been lost. Both cases are associated with unusual reproductive behavior, including male pregnancy and sexual parasitism. Several cold water fish in the Atlantic cod (Gadinae) family carry an aicda gene that encodes for a full-length enzyme but lack affinity-matured antibodies and rely on antibodies of broad antigenic specificity. Hence, we examined the functionality of their AID. Results By combining genomics, transcriptomics, immune responsiveness, and functional enzymology of AID from 36 extant species, we demonstrate that AID of that Atlantic cod and related fish have extremely lethargic or no catalytic activity. Through ancestral reconstruction and functional enzymology of 71 AID enzymes, we show that this enzymatic inactivation likely took place relatively recently at the emergence of the true cod family (Gadidae) from their ancestral Gadiformes order. We show that this AID inactivation is not only concordant with the previously shown loss of key adaptive immune genes and expansion of innate and cell-based immune genes in the Gadiformes but is further reflected in the genomes of these fish in the form of loss of AID-favored sequence motifs in their immunoglobulin variable region genes. Conclusions Recent demonstrations of the loss of the aicda gene in two fish species challenge the paradigm that AID-driven secondary antibody diversification is absolutely conserved in jawed vertebrates. These species have unusual reproductive behaviors forming an evolutionary pressure for a certain loss of immunity to avoid tissue rejection. We report here an instance of catalytic inactivation and functional loss of AID rather than gene loss in a conventionally reproducing vertebrate. Our data suggest that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment relieved the pressure to maintain robust secondary antibody diversification. We suggest that in this unique scenario, the AID-mediated collateral genome-wide damage would form an evolutionary pressure to lose AID function.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

“…The absence of MHCII and CD4 and the inactivation of the AID gene [ 30 ] in Atlantic cod provides strong evidence against the presence of somatic hypermutation in this species. Consequently, it relies exclusively on deletions, and P- and N nucleotide additions and the combinatorial variability of its VDJ genes to diversify its immunoglobulin sequences.…”

“…It is a prolific species in the Atlantic, despite its inconsistent specific antibody response to antigens in experimental settings [ 28 ]. The Atlantic cod lacks MHCII, invariant chain, and has a truncated CD4 [ 29 ], in conjunction with loss of function of the AID protein [ 30 ] that mediates somatic hypermutation. This genomic architecture is expected to yield a constrained antibody repertoire diversity.…”

“…The genome analysis revealed not only the absence of MHCII but also the lack of the invariant chain and a truncated, non-functional CD4, implying a compromised T cell-dependent antibody response pathway. Additionally, the AID protein in Atlantic cod was shown to be inactive [ 30 ]. The complete lack of the MHCII-CD4 pathway as well as the absence of AID activity preclude the occurrence of somatic hypermutation and affinity maturation in Atlantic cod.…”

A survey of the adaptive immune genes of the polka-dot batfish Ogcocephalus cubifrons

“…Low-level aicda expression was also detected in RNA-seq libraries (Table 1 ). Some fish species, such as the Atlantic cod, have intact aicda genes which nevertheless encode AID proteins with little to no catalytic activity [ 16 ]. As our sequencing data lacked sufficient coverage to conclusively demonstrate SHM in immunoglobulin sequences, we instead assessed catalytic potential by aligning key regions of the O. cubifrons AID protein with a representative set of high (human, zebrafish and pufferfish) and low (atlantic cod) activity AID orthologs.…”

450 million years in the making: mapping the evolutionary foundations of germinal centers

“…demonstrate a loss of both MHC class II and CD4 genes ( 116 , 117 ), nullifying canonical T cell dependent antibody responses in this group. Additionally, cod AICDA is catalytically inactive, severely limiting secondary antibody diversification ( 118 ). The evolution of unique reproductive strategies also appears to have influenced the loss of immune genes in other bony fishes.…”