Functional Analysis of PAAN-AG5, the Baboon Counterpart of HLA-G5

PAAN-AG5（HLA-G5 的狒狒对应物）的功能分析

• 批准号: 7813884
• 负责人: DAUDI K LANGAT
• 金额: $ 8.17万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7813884
• 关键词: Address; Allogenic; Alternative Splicing; Animal Model; B-Lymphocytes; Bacteria; CD8-Positive T-Lymphocytes; CD8B1 gene; Cells; Characteristics; Conceptions; Data; Development; Embryo; Embryonic Development; Ensure; Ethics; Failure; Fertilization; Fetus; GTP-Binding Proteins; Gametogenesis; Genes; Genetic Polymorphism; Goals; HLA Antigens; HLA-G5; Heart Transplantation; Human; Immune; Immune response; Immune system; Immunosuppressive Agents; Laboratories; Leukocytes; Maternal-Fetal Exchange; Membrane; Messenger RNA; Modeling; Natural Killer Cells; Papio; Papio anubis; Pathology; Pathway interactions; Phenotype; Placenta; Placentation; Play; Pre-Eclampsia; Pregnancy; Premature Labor; Protein Isoforms; Proteins; Public Health; RNA Processing; Recombinants; Reproductive system; Research; Role; T-Lymphocyte; Techniques; Testing; Therapeutic; Transcript; Transgenic Mice; Transplantation; Villous; base; cytotrophoblast; design; implantation; in vitro Model; in vivo; in vivo Model; insight; kidney cell; macrophage; monocyte; peripheral blood; pre-clinical; programs; protein expression; public health relevance; research study; trophoblast; tumor; tumor progression

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the mechanisms by which rejection of the semiallogeneic fetus by the maternal immune system is avoided. The fetus actively participates in its own protection by expression of the class Ib human leukocyte antigen-G (HLA-G), on trophoblast cells during pregnancy. HLA-G is thought to be critical in survival of the semi-allogenic fetus; it programs immune cells at the maternal-fetal interface into immunosuppressive phenotypes. Definitive proof of HLA-G function remains elusive since in vivo experiments in humans are not possible due to ethical concerns. We have been assessing the suitability of using the olive baboon (Papio anubis) as a model for HLA-G in vivo studies. In pursuit of this goal, we identified a unique HLA-G-like MHC class Ib gene termed Paan-AG in the baboon. HLA-G and Paan-AG display remarkable similarity in the RNA processing, including alternative splicing of the mRNA, resulting in transcripts that encode membrane-anchored (HLA-G1-4 or Paan-AG1-4) and soluble proteins (HLA-G5-7 or Paan-AG5 respectively). In humans, HLA-G5 is emerging as one of the critical isoforms important in pregnancy, transplantation and cancer progression. Both HLA-G5 and the baboon counterpart, Paan-AG5, are highly expressed by villous and extravillous cytotrophoblast cells in the human and baboon placenta respectively [1,4,6]. Based on these observations, our hypothesis is that baboon placental Paan-AG5, as with human placental HLA-G5, drives the maternal immune response into pathways beneficial to pregnancy. To test this hypothesis, it is necessary to first establish the effects of Paan-AG5 protein on immune cells for comparison with HLA-G5 effects. HLA-G5 interacts with all major subsets of immune cells, including natural killer (NK) cells, CD4+ and CD8+ T-lymphocytes, B-lymphocytes, macrophages and dentritic cells. The planned experiments are designed to assess whether recombinant Paan-AG5 acts similarly on sub- sets of immune cells purified from baboon peripheral blood leukocytes. The specific aims of this proposal are to (i) generate and characterize recombinant Paan-AG5 protein, and (ii) assess the ability of Paan- AG5 to act as an immune suppressor molecule for the benefit of pregnancy. Techniques established in our laboratory will be used to generate recombinant prokaryotic Paan-AG5 in bacteria and eukaryotic FLAG- tagged Paan-AG5 in human embryonic kidney cells (HEK293). We will assess effects of the purified proteins on natural killer (NK) cells, CD4+ and CD8+ T-cells, monocytes, macrophages and dentritic cells. The results of this study may provide critical data on the potential function of Paan-AG5 (and by inference, HLA-G5) in pregnancy. Elucidation of the role of HLA-G in pregnancy is essential as a prerequisite to assessing the therapeutic potential of recombinant HLA-G proteins in pregnancy-related pathologies and transplantation. These studies may also provide important information regarding the suitability of the olive baboon as a model for in vivo HLA-G functional studies to assess the therapeutic potential of recombinant HLA-G5. PUBLIC HEALTH RELEVANCE: The proposed study is relevant to public health because inappropriate expression of HLA-G5 and/or HLA-G6 in humans has been associated with difficulties in conception or pregnancy failure due to preterm labor, preeclampsia or other pregnancy pathologies. This suggests that recombinant isoforms of these proteins may be of value in designing therapeutic strategies to address these pregnancy problems. An animal model with a reproductive system similar to that of humans, such as the olive baboon, is essential in order to perform pre- clinical tests to assess therapeutic applications of HLA-G. This proposal is aimed at assessing the feasibility of using the olive baboon as a model for HLA-G in vivo experiments.

描述（由申请人提供）：这项研究的长期目标是阐明避免母体免疫系统拒绝分子胎儿的机制。胎儿在​​怀孕期间通过表达IB人白细胞抗原G（HLA-G）的表达来积极参与其自身保护。 HLA-G被认为对半生殖胎儿的生存至关重要。它将母体界面的免疫细胞编程为免疫抑制表型。 HLA-G功能的确定性证明仍然难以捉摸，因为由于道德问题，无法进行人体体内实验。我们一直在评估使用橄榄狒狒（Papio Anubis）作为HLA-G体内研究模型的适用性。为了实现这一目标，我们确定了一个独特的HLA-G型MHC类IB基因在狒狒中称为Paan-Ag。 HLA-G和PAAN-AG在RNA处理中显示出显着的相似性，包括mRNA的替代剪接，导致编码膜锚定的转录本（HLA-G1-4或PAAN-AG1-4）和可溶性蛋白（分别为HLA-G5-7或PAAN-AG5）。在人类中，HLA-G5正在成为怀孕，移植和癌症进展中重要的关键同工型之一。 HLA-G5和狒狒对应物Paan-Ag5均通过人和狒狒胎盘中的绒毛和跨性细胞粒细胞细胞高度表达[1,4,6]。基于这些观察结果，我们的假设是狒狒胎盘paan-ag5与人胎盘HLA-G5一样，将母体免疫反应驱动到有益于怀孕的途径中。为了检验这一假设，有必要首先建立PAAN-AG5蛋白对免疫细胞的影响，以与HLA-G5效应进行比较。 HLA-G5与免疫细胞的所有主要子集相互作用，包括天然杀伤（NK）细胞，CD4+和CD8+ T淋巴细胞，B-淋巴细胞，巨噬细胞和硬质细胞。计划的实验旨在评估重组PAAN-AG5是否在从狒狒外周血白细胞纯净的免疫细胞的亚组中起作用。该提案的具体目的是（i）产生和表征重组PAAN-AG5蛋白，（ii）评估Paang5充当免疫抑制分子的能力，从而使妊娠受益。在我们的实验室中建立的技术将用于在人类胚胎肾细胞中生成细菌中的重组核Paan-Ag5和真核旗标记的PAAN-AG5（HEK293）。我们将评估纯化的蛋白质对天然杀伤（NK）细胞，CD4+和CD8+ T细胞，单核细胞，巨噬细胞和硬质细胞的影响。这项研究的结果可能会提供有关妊娠中Paan-AG5（以及推断）的潜在功能的关键数据。阐明HLA-G在妊娠中的作用是评估重组HLA-G蛋白在妊娠相关病理和移植中的治疗潜力的必要条件。这些研究还可能提供有关橄榄狒狒作为体内HLA-G功能研究模型的适用性的重要信息，以评估重组HLA-G5的治疗潜力。 公共卫生相关性：拟议的研究与公共卫生有关，因为人类中HLA-G5和/或HLA-G6的不当表达与由于早产劳动，前宾夕法尼亚先兆子痫或其他怀孕病理学而导致的构思或妊娠失败的困难有关。这表明这些蛋白质的重组同工型在设计治疗策略以解决这些妊娠问题可能具有价值。具有类似于人类的生殖系统的动物模型，例如橄榄狒狒，对于进行临床测试以评估HLA-G的治疗应用至关重要。该建议旨在评估使用橄榄狒狒作为HLA-G体内实验模型的可行性。