Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single-stranded DNA binding.

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.

The cellular context of AID expressing cells in fish lymphoid tissues.

It has long been held that the cold-blooded vertebrates lack mammalian-like germinal centers, though they do have affinity maturation and the immunoglobulin mutator activation-induced cytidine deaminase or AID. Using AID as a marker of sites of somatic hypermutation, we have identified discrete cell clusters of up to several thousand cells, in the spleen and kidney of channel catfish (Ictalurus punctatus), which may be primordial germinal centers. In situ hybridization revealed that AID expressing cells are interspersed or surrounded by a population of pigmented CSF1-R expressing cells called melano-macrophages. Significantly, melano-macrophages or associated reticular cells have been previously noted for their ability to retain soluble antigen on or near their surface for several weeks following vaccination. Laser capture microdissection and RT-PCR were used to establish that these cell clusters also contained cells expressing Ig heavy chain transcripts as well as transcripts of TcRbeta and the putative CD4 homologue of fish. These observations, coupled with past work showing that mutations develop in B-cell lineages in fishes, allow us to develop a model for how affinity maturation may have evolved in early gnathostome vertebrates.

Cloning and expression of the AID gene in the channel catfish.

A full-length activation-induced cytidine deaminase (AID or Aicda) cDNA has been obtained from the channel catfish (Ictalurus punctatus). A single open reading frame predicts a 209 amino acid protein that has 57% identity and 73% similarity with the AID proteins of mouse and human. All residues that have previously been found to be critical for deamination, as well as for somatic hypermutation, are conserved in the catfish AID. These residues are also conserved in AID proteins predicted, from genome database sequences, to be expressed in Fugu and zebrafish. The catfish AID is expressed at low levels in spleen, kidney, intestine and fin margins, but not in muscle, liver or brain. Immunoglobulin heavy chain (IgH) is also expressed in the tissues where AID is expressed. The 'ectopic' expression of AID in non-lymphoid tissue was unexpected and not readily explained. However, the identification of a fish AID gene will allow us to determine the tissue architecture and locations for affinity maturation in fish.