AID and Its Impact on Antibody Genetic Alteration

Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. M. Muramatsu et al., "Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme," Cell, 102:553-63, Sept.1, 2000. (Cited in 134 papers) P. Revy et al., "Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2)," Cell, 102:565-75, Sept. 1, 2000. (Cited in 111 papers) Antibodies undergo three phases of genetic alteration. Researchers know that perhaps one billion different specificities of human antibodies are created from just 1,000 genes in the germline by shifting and reshifting various segments, called variability (V), diversity (D), and joining (J). These antibodies together recognize nearly every pathogen and toxin the body could encounter. Researchers know, too, that an antibody's affinity for its antigen tends to increase over the course of a response--a phenomenon known as affinity maturation--through a process called somatic hypermutation. SHM takes place in the lymph node germinal centers as B cells compete for limited antigen, with the ability to survive conferred only on those with the highest affinity. A third genetic process, which also occurs in the germinal centers, is class switch recombination (CSR). In CSR, the molecule's constant (C) segment--encoding the stem of the Y-shaped antibody--is changed, conferring different functions on the antibody. Coauthored by Tasuko Honjo, these two Hot Papers1,2 show that a gene called AID (activation-induced cytidine deaminase) regulates SHM and CSR in both humans and mice. "As far as I'm concerned, those papers opened up the whole area," says CSR investigator Matthew Scharff, Albert Einstein College of Medicine. "They were seminal papers." They showed that "the gene and the protein it encoded were necessary both for isotype switching and for the somatic mutation of antibody genes." Michael Neuberger, a Medical Research Council researcher in Cambridge who also works on CSR, agrees: "It was obvious when the papers came out at the time that important genes had been identified, which were either involved directly in the processes, or might take lymphocytes to a stage of development where they could do these processes.... It was, in fact, what allowed the field to move forward in a quantum jump." The papers' impact was greatly enhanced by the finding that both mice and humans show the same effect. "It gave the research that much more credence," says SHM investigator Patricia Gearhart, National Institutes of Health. WHAT MAKES IT HAPPEN The recombination activating genes RAG-1 and RAG-2 were isolated in 1989 and 1990.3,4 These are the only lymphoid-specific genes required for V(D)J recombination. Discovery of the RAG genes was the first step to understanding the processes involved in this profound transformation of lymphocyte genetic material. Reprinted with permission from Nature AID INDUCEMENT: Hybridomas N89 (nonsense in leader) and N114 (nonsense in V region) were transfected with either an empty vector or the hAID construct. The frequency of nonsense revertants, as detected with the ELISA-spot assay, is plotted. Typical ELISA spots for N89 are shown in the inset. The difference in the frequencies or reversion of N114 vector and N114 hAID was statistically significant (P<0.05). (Nature, 45:802-6, 2002) But RAGs do not seem to play a role in SHM or CSR, which is not surprising. "V(D)J recombination is entirely different from class switch recombination," Honjo says. RAG recognizes a given sequence of DNA, but the target for switch recombination is somewhat vague, he explains. And, somatic hypermutation takes place on the variable region of the immunoglobin gene, creating point mutations. It was equally unlikely that SHM and CSR would be related to each other. But, it turns out, they are. DISCOVERY OF AID Honjo has studied the mechanism of class switching for 25 years. His Kyoto University group had developed a system in which a transgenic substrate in cultured murine lymphoma cells were induced to switch from IgM production to IgA production upon stimulation. By comparing cDNA from stimulated and unstimulated cells, they identified a novel homolog of the RNA editing gene, APOBEC1, which is specifically expressed in germinal-center B cells.5 When the researchers overexpressed their newly discovered activation-induced cytidine deaminase in lymphoma cells, the transgene underwent class switching in the absence of external stimulation. CSR was completely blocked in cells from chimeric and transgenic mice lacking the AID gene.1 This, perhaps, was expected. But what was not expected was that AID-deficient cells were also lacking in somatic hypermutation. "People never thought they were regulated by a single protein or a single gene," Honjo says. "It was really surprising to the field, and we were also surprised." AND IN HUMANS, TOO At the same time, Anne Durandy's INSERM group in Paris had been investigating a cohort of patients with a rare form of hyper-IgM syndrome (HIGM). The researchers had ruled out defects such as faulty cytokine or receptor genes previously shown to cause HIGM in other patients. Instead, in addition to their B cells being unable to undergo CSR, these patients displayed a phenotype similar to AID-deficient mice: They lacked SHM and had enlarged lymph nodes due to giant germinal centers. Durandy collaborated with the Japanese group. They discovered deleterious mutations in these patients, and they mapped the huAID gene to the same chromosomal location as its murine counterpart.2 Courtesy of Svend Petersen-Mahrt, Michael Neuberger AID ENLISTMENT: Some researchers think that recruiting AID to the immuno-globulin locus causes localized deamination of dC-dU. Consequently, the repair of this uracil in DNA leads to SHM, GC or CSR, depending on the pathway used for resolving this dU lesion, as well as the precise location--either the IgV domain or in the vicinity of the switch region. THE KNOWN AND UNKNOWN Based on its homology to other proteins, some scientists believe that AID encodes a cytidine deaminase, converting cytidine to uradine. But less consensus exists on what the substrate is, as well as where AID operates in the pathways leading to CSR and SHM. Neuberger showed that AID can deaminate DNA at cytidine in Escherichia coli,6 as can APOBEC1.7 Together, these experiments and others are "most clearly and easily understood by saying [AID] acts directly on DNA," says Scharff. Honjo does not see it quite this way. He wrote in Proceedings of the National Academy of Sciences last month that AID "might be an editing enzyme of a preexisting mRNA precursor, and the edited mRNA may code for an enzyme involved in CSR and SHM."8 He has been championing an RNA-editing model since he discovered AID's homology with APOBEC1. But Honjo may be "a lone tree standing there at this point," Gearhart says. He was the sole scientist interviewed who did not lean toward a DNA-editing model. "I think everyone at [the] Keystone [Symposium in January] was talking along the DNA deamination theory lines," she adds. Whether it is a DNA- or RNA-editing enzyme, AID is known to be the only B cell-specific factor necessary for initiating CSR and SHM. Scharff's group has demonstrated that expression of AID in cells is sufficient to SHM in the nonlymphoid ovary cells of the Chinese hamster,9 and Honjo's group has shown it to be sufficient for CSR in fibroblasts.10 "That said that AID was probably the only B cell- specific protein that was required, and that the other molecules that were involved were molecules that were ubiquitously expressed in all sorts of other cells," Scharff explains. Meanwhile, Durandy's group has shown that in patients deficient in AID, the CSR-induced DNA breaks do not occur, "thus placing AID upstream of the DNA breaks required for class switch recombination (and likely for SHM)," she comments. Says Scharff: While most people believe AID is acting directly on DNA, no real biochemical evidence has been published demonstrating that, or that AID is found in the nucleus, where it would have to be to reach DNA. Scientists are now trying to work out the biochemistry of AID: what its target is, how it is regulated, how it gets its specificity, whether other factors are necessary to chaperone it, and the like. Besides the intrinsic appeal of understanding a system's basic biology, knowing how AID functions may someday help other areas of research, including lymphomas and other cancers, allergies, and other immunological disorders. And, citing his work that AID overexpression can alter other genes as well,11 Scharff speculates that research may even "lead to a routine to make better monoclonal antibodies." Josh P. Roberts (tcwriter@msn.com) is a freelance writer in Minneapolis, Minn. References 1. M. Muramatsu et al., "Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme," Cell, 102:553-63, Sept.1, 2000. (Cited in 134 papers) 2. P. Revy et al., "Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2)," Cell, 102:565-75, Sept. 1, 2000. (Cited in 111 papers). 3. D.G. Schatz et al., "The V(D)J recombination activating gene, RAG-1," Cell, 59:1035-48, 1989. 4. M.A. Oettinger et al., "RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination," Science, 248:1517-23, 1990. 5. M. Muramatsu et al., "Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells," J Biol Chem, 274:18470-6, 1999. 6. S.K. Petersen-Mahrt et al., "AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification," Nature, 418:99-103, 2002. 7. R.S. Harris et al., "RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators," Molec Cell, 10:1247-53, November 2002. 8. T. Doi et al., "De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination," Proc Natl Acad Sci, Feb. 18, 2003, 10.1073/pnas.0437710100. 9. A. Martin, M.D. Scharff, "Somatic hypermutation of the AID transgene in B and non-B cells," Proc Natl Acad Sci, 99:12304-8, Sept. 17, 2002. 10. K. Yoshikawa et al., "AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts," Science, 296:2033-6, 2002. 11. A. Martin et al., "Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas," Nature, 415:802-6, 2002.

AID and Its Impact on Antibody Genetic Alteration

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