EAGER: Functional Study of the Lamprey AID/APOBEC Family of Cytidine Deaminases

无颌脊椎动物（七鳃鳗和盲鳗）的免疫基因多样化的过程与有颌脊椎动物的适应性免疫系统不同。pi已经获得证据表明，胞苷脱氨酶（CDAs）的AID/APOBEC家族可能有助于使有颌和无颌脊椎动物的受体多样化。拟议的研究将确定七鳃鳗中显著多样化的CDA家族的功能潜力。这些研究将提供深入了解其抗原受体的体细胞多样化的机制。这种对七鳃鳗胞苷脱氨酶的探索性功能分析符合EAGER提案，因为这种分析将侧重于高风险-高回报的研究：高风险-其他分子参与VLR基因组装和亲和成熟的可能性；高回报（变革性）-抗原受体介导的适应性免疫系统的新范式，以及VLR分子的新抗体工程和治疗潜力。此外，培养STEM学科的本科生和监督患有自闭症谱系障碍的高中生将以多种方式造福社会。pi假设无颌脊椎动物利用AID/APOBEC家族成员触发VLR基因组装：CDA1和CDA2可能分别参与VLRA/VLRC和VLRB组装。该实验室现在已经确定了另外三个类似cda1的基因（CDA3、CDA4和CDA5）。此外，通过对七鳃鳗淋巴细胞样细胞的转录分析，他们发现了四种CDA2剪接变体，这一分析可能解释了为什么以前报道的CDA2异构体没有证明胞苷脱氨酶的活性。这项对七鳃鳗AID/APOBEC家族CDAs的功能研究的预期结果将极大地促进我们对无颌脊椎动物中抗原受体基因的体细胞组装的了解。甚至不是所有的人类AID/APOBEC家族成员都被很好地表征，在大多数基础脊椎动物代表中提出的研究可能有助于对人类CDA家族的分析。该研究结果还可能通过优化七鳃鳗cdna的诱变，提高工程七鳃鳗VLR抗体的亲和力，该抗体在人类中具有重要的诊断和治疗潜力。总之，来自七鳃鳗cda分析的结果将为无颌和有颌脊椎动物的适应性免疫系统范式提供重要的拼图。

The jawless vertebrates (lampreys and hagfish) diversify their immune genes through a different process than the adaptive immune system in jawed vertebrates. The PIs have obtained evidence that the AID/APOBEC family of cytidine deaminases (CDAs) may contribute to diversify receptors in both jawed and jawless vertebrates. The proposed studies will determine the functional potential of the remarkably diverse CDA family in lampreys. These studies will provide insight into the mechanisms involved in the somatic diversification of their antigen receptors. This exploratory functional analysis of the lamprey cytidine deaminases fits with the EAGER proposal because this analysis will focus on high risk-high reward study: High risk- Possibility of involvement of other molecules for VLR gene assemblies and affinity maturations; High reward (transformative)- New paradigm for antigen receptor-mediated adaptive immune systems, and novel antibody engineering and therapeutic potential of VLR molecules. In addition, training of undergraduate students in STEM disciplines and supervising high school students with autism spectrum disorder will benefit society in diverse ways. The PIs hypothesize that jawless vertebrates use AID/APOBEC family members to trigger VLR gene assembly: CDA1 and CDA2 may participate in VLRA/VLRC and VLRB assembly, respectively. The lab has now identified three additional CDA1-like genes (CDA3, CDA4 and CDA5). In addition, through transcriptional analysis of lymphocyte-like cells in lampreys, they have identified four CDA2 splice variants, the analysis of which probably explain why cytidine deaminase activity was not demonstrable for the previously reported CDA2 isoform. The expected results from this functional study of the lamprey AID/APOBEC family of CDAs will significantly advance our knowledge of how somatic assembly of the antigen receptor genes occur in jawless vertebrates. Not even all of the human AID/APOBEC family members have yet been well characterized, and the proposed research in the most basal vertebrate representatives may facilitate analysis of this CDA family in humans. The consequences of this proposal may also lead to improvement of the affinity of engineered lamprey VLR antibodies, which have important diagnostic and therapeutic potential in humans, by optimizing mutagenesis with lamprey CDAs. Collectively, the results derived from the analysis of lamprey CDAs will provide an important piece of the puzzle to the paradigm of adaptive immune systems in jawless and jawed vertebrates.