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: 7659319
• 负责人: DAUDI K LANGAT
• 金额: $ 7.5万
• 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/7659319
• 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）：本研究的长期目标是阐明避免母体免疫系统排斥半同种异体胎儿的机制。妊娠期间，胎儿通过在滋养层细胞上表达Ib类人类白细胞抗原-G（HLA-G）积极参与自身保护。HLA-G被认为是半同种异体胎儿存活的关键;它将母胎界面的免疫细胞编程为免疫抑制表型。HLA-G功能的连续性证据仍然难以捉摸，因为由于伦理问题，在人体中进行体内实验是不可能的。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。在追求这一目标，我们确定了一个独特的HLA-G样的MHC Ib类基因称为Paan-AG在狒狒。HLA-G和Paan-AG在RNA加工中显示出显著的相似性，包括mRNA的选择性剪接，导致编码膜锚定蛋白（HLA-G1-4或Paan-AG 1 -4）和可溶性蛋白（分别为HLA-G5-7或Paan-AG 5）的转录物。在人类中，HLA-G5正在成为妊娠、移植和癌症进展中重要的关键同种型之一。HLA-G5和狒狒对应物Paan-AG 5分别在人和狒狒胎盘的绒毛和绒毛外细胞滋养层细胞中高度表达[1，4，6]。基于这些观察，我们的假设是狒狒胎盘Paan-AG 5，与人类胎盘HLA-G5一样，驱动母体免疫应答进入有利于妊娠的途径。为了验证这一假设，有必要首先确定Paan-AG 5蛋白对免疫细胞的影响，以与HLA-G5的影响进行比较。HLA-G5与免疫细胞的所有主要亚群相互作用，包括自然杀伤（NK）细胞、CD 4+和CD 8 + T淋巴细胞、B淋巴细胞、巨噬细胞和树突细胞。设计计划的实验以评估重组Paan-AG 5是否对从狒狒外周血白细胞纯化的免疫细胞亚组起类似作用。该提议的具体目的是（i）产生和表征重组Paan-AG 5蛋白，和（ii）评估Paan-AG 5作为免疫抑制分子用于妊娠益处的能力。我们实验室建立的技术将用于在细菌中产生重组原核Paan-AG 5和在人胚肾细胞（HEK 293）中产生真核FLAG标记的Paan-AG 5。我们将评估纯化的蛋白质对自然杀伤（NK）细胞、CD 4+和CD 8 + T细胞、单核细胞、巨噬细胞和树突状细胞的作用。这项研究的结果可能提供关键数据的潜在功能Paan-AG 5（和推断，HLA-G5）在怀孕。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先决条件。这些研究也可能提供重要的信息，橄榄狒狒作为体内HLA-G功能研究的模型，以评估重组HLA-G5的治疗潜力的适用性。 公共卫生相关性：所提出的研究与公共卫生相关，因为人类HLA-G5和/或HLA-G6的不适当表达与由于早产、先兆子痫或其他妊娠病理学导致的受孕困难或妊娠失败相关。这表明，这些蛋白质的重组异构体可能是有价值的设计治疗策略，以解决这些妊娠问题。为了进行临床前测试以评估HLA-G的治疗应用，具有与人类类似的生殖系统的动物模型（例如橄榄狒狒）是必不可少的。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。