Activation-induced deaminase cloning, localization, and protein extraction from young V_H-mutant rabbit appendix

Abstract

Studies in mouse, human, and chicken suggest that activationinduced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics. mass spectrometry ͉ gene conversion ͉ somatic hypermutation T he rabbit is an excellent animal model for study of somatic hypermutation (SHM) and gene conversion (GC) of rearranged variable regions (V) of Ab heavy (H) and light (L) chain genes. The appendix of neonatal rabbits is a critical site for development of the primary Ab repertoire (1, 2). Rearrangements of H and L chain genes occur in sites including fetal liver, omentum, and bone marrow. After birth, B cells with rearranged genes migrate into gut-associated lymphoid tissue such as the appendix. Gut microflora are required for B cells to proliferate and develop in appendix follicles (3). At Ϸ3 wk of age, diversification of rearranged gene sequences by GC and SHM starts within the appendix-follicle microenvironment (2). GC diversifies sequences encoding V regions of H and L chains through introduction of sequences from upstream donor V gene segments. B cells that have undergone class-switch recombination (CSR), particularly to IgA, are also found in rabbit appendix at Ϸ3 wk of age (2). Activation-induced deaminase (AID) is involved in three processes of Ab diversification and maturation, SHM, CSR (4-7), and GC (8,9), that all coexist in young rabbit appendix. Thus far, the only published evidence for the role of AID in GC comes from two studies showing that in the chicken DT40 bursal tumor cell line that carries out GC in vitro, no GC occurs when AID is knocked out on both chromosomes (8, 9). Although there are several hypotheses about how AID functions, data are accumulating that suggest that AID deaminates cytosine to uracil on the non-template strand of DNA during transcription (10). It is also possible that AID deaminates cytidine to uridine on an unknown mRNA (11,12). Data are still emerging that suggest AID has different roles and cofactor interactions when participating in CSR and SHM (13,14).We used 1-to 6-wk-old ali͞ali V H mutant rabbits to study rabbit AID. These mutants have a small (Ϸ10 kb) deletion encompas...

Active nuclear import and cytoplasmic retention of activation-induced deaminase

“…AID dimerization through the β2 strand has been proposed based on the structure of A2 33 . We tested the effect of perturbing the predicted AID β2, residues [40][41][42][43][44][45][46][47][48][49][50][51][52][53] in our model, on oligomerization by introducing one (F46A), two (F46A/Y48A) 33 or four (F46A/Y48A/R50G/N51A, named AID FYRN) mutations. The ability of each of these mutants to interact with wt AID was monitored by comparing their efficiency in coimmunoprecipitating with AID-Flag.…”

“…The stoichiometry and architecture of the biologically relevant AID molecule is unknown but many indirect evidences suggest that it will have quaternary structure 33,35,42,47,48 as all cytidine deaminases including the APOBECs do 33,49,50 . Predicting AID dimerization through β2 (ref.…”

“…The difference with AID-GFP, which accumulates in the nucleus of Ramos, could be explained by a combination of overexpression and the C-terminal tag weakening retention, just as an N-terminal tag affects import. AID is vastly confined to the cytoplasm of germinal center B cells 48,[53][54][55] . Only a very small proportion of the B cells in rabbit appendix 48 and human tonsils 53 , and hardly any cell in unsynchronized populations overexpressing AID-GFP, seem to contain predominantly nuclear AID.…”

“…AID is vastly confined to the cytoplasm of germinal center B cells 48,[53][54][55] . Only a very small proportion of the B cells in rabbit appendix 48 and human tonsils 53 , and hardly any cell in unsynchronized populations overexpressing AID-GFP, seem to contain predominantly nuclear AID. In the light of our results it seems unlikely that such accumulation might be cell cycle regulated or has much physiological significance.…”

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

“…The rabbit's value to biomedical research includes its use in the production of high quality antiserum, studies of immunoglobulin structure and regulation (Kindt, 1975), B cell development (Yang et al, 2005;Sinha et al, 2006), and antibody repertoire development (Rhee et al, 2004;Rhee et al, 2005), and as models of inflammation and infectious diseases. In the study of syphilis, the rabbit is the preferred experimental model because it develops clinical manifestations that closely resemble human disease (Turner and Hollander, 1957).…”