SOLUBLE ISOFORMS OF HLA-G: STRUCTURE, REGULATION AND FUNCTION

摘要--项目I 成功的人类妊娠被认为依赖于滋养层细胞产生人类白细胞抗原G蛋白。 胎盘。人类白细胞抗原-G基因产生多个转录本，编码四个膜蛋白和三个可溶性蛋白 蛋白质。两种可溶性亚型，即人类白细胞抗原-G5(G5)和人类白细胞抗原-G6(G6)(也称为sHLA-G1和SHLA-G2， 分别)，不仅存在于母胎界面，而且在母体血液中循环 怀孕了。这些蛋白质似乎具有重要的生物学意义：科学文献记录了它们之间的联系 妊娠成功和体外培养胚胎培养上清液中高水平的可溶性人类白细胞抗原-G，以及 我们实验室的关键初步实验表明，反复自然流产的妇女不会 妊娠期仅不能提高其血清中的HLA-G5水平，而可能降低其血清中的HLAG6水平。为了 研究这些蛋白质对母亲对基因不同的胚胎/胎儿的免疫反应的影响， 我们开发了真核表达载体，并产生了针对这些蛋白的单抗。 使用这些独特试剂的研究表明，G5和G6在初级和次级方面不同 结构。表达研究记录了不同亚型在其定位上的显著差异。 对于特定的滋养层细胞亚群，表达调控的实验已经证明 一般的和特定于异构体的调节条件，以及对功能的研究已经确定了定性和 两种异构体之间的数量差异。在本申请中，我们提出的研究将扩展我们的 这些强大的妊娠相关蛋白的知识。AIM 1旨在调查潜在的 配基：单核巨噬细胞中的受体相互作用，定位表达，并进行结合研究。 AIM 2的目标是建立调控G5和G6蛋白差异表达的机制。 妊娠早期和晚期胎盘滋养细胞亚群。在AIM 3中，目的是 研究G5和G6如何编程单核吞噬细胞。我们将系统地比较 足月胎盘样本和年龄匹配的正常胎盘样本的结果 来自问题妊娠的样本，即先兆子痫和早产/分娩，并有和没有感染。 这项拟议的研究与公共健康的相关性取决于这样一个事实，即至少十分之一的夫妇 经历过生育困难，有很高比例的妊娠失败是由于早产或非早产 感染了。一个潜在的原因可能是G5/G6或其合成或表达的异常 LILRB1/LilrB2受体。我们希望在这里提供对设计至关重要的全新信息 治疗策略，以解决人类白细胞抗原-G相关的妊娠病理。

ABSTRACT - PROJECT I Successful human pregnancy is believed to rely upon production of HLA-G proteins by trophoblast cells in placentas. The HLA-G gene generates multiple transcripts that encode four membrane and three soluble proteins. Two of the soluble isoforms, HLA-G5 (G5) and HLA-G6 (G6) (also known as sHLA-G1 and SHLA-G2, respectively), are not only present at the maternal-fetal interface but also circulate in maternal blood throughout pregnancy. These proteins appear to be biologically important: the scientific literature documents associations between pregnancy success and high levels of soluble HLA-G in the supernatant culture media of in vitro cultured embryos, and critical preliminary experiments in our laboratory indicate that women who suffer recurrent spontaneous abortions not only fail to increase their serum levels of HLA-G5 but may decrease their levels of HLA-G6 with pregnancy . In order to study the impact of these proteins on the mother's immune responses to her genetically different embryo/fetus, we developed eukaryotic expression vectors and generated monoclonal antibodies specific for the proteins. Studies using these unique reagents have demonstrated that G5 and G6 differ in primary and secondary structures. Expression studies have documented marked differences between the isoforms in their localization to specific subpopulations of trophoblast cells, experiments on regulation of expression have demonstrated both general and isoform-specific regulatory conditions, and studies on function have identified qualitative and quantitative differences between the two isoforms. In this application we propose studies that will expand our knowledge of these powerful, pregnancy-associated proteins. AIM 1 is designed to investigate potential ligand:receptor interactions in mononuclear phagocytes, mapping expression, and performing binding studies. The goal of AIM 2 is to establish mechanisms of regulation of differential expression of G5 and G6 proteins in subpopulations of trophoblast cells from early and late gestation placentas. In AIM 3 the purpose is to investigate how G5 and G6 function to program mononuclear phagocytes. We will systematically compare results on samples from 1st trimester with term placentas and results on normal placentas with age-matched samples from problem pregnancies, i.e., preeclampsia and preterm labor/delivery with and without infection. The relevance of the proposed research to public health rests on the fact that at least one in ten couples experiences fertility difficulties and a high proportion of pregnancies fail due to preterm labor associated or not with infection. One underlying cause may be aberrancies of synthesis or expression of G5/G6 or their LILRB1/LILRB2 receptors. We expect here to provide entirely novel information that is essential to the design of therapeutic strategies to address HLA-G-associated pathologies of pregnancy.