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

摘要-项目I 人类成功怀孕被认为依赖于胎盘滋养层细胞产生HLA-G蛋白， 胎盘HLA-G基因产生多种转录本，编码四种膜和三种可溶性蛋白。 proteins.两种可溶性同种型，HLA-G5（G5）和HLA-G6（G6）（也称为sHLA-G1和SHLA-G2， 不仅存在于母胎界面，而且还在整个母体血液中循环 怀孕这些蛋白质在生物学上似乎很重要：科学文献记录了 妊娠成功和体外培养胚胎的上清液培养基中高水平的可溶性HLA-G，以及 我们实验室的关键性初步实验表明，反复自然流产的妇女 仅不能提高其血清HLA-G5水平，但可降低其HLA-G6水平。为了 研究这些蛋白质对母亲对遗传不同的胚胎/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对这些蛋白质特异的单克隆抗体。 使用这些独特试剂的研究表明，G5和G6在原发性和继发性 结构.表达研究已经证明了异构体在其定位上的显著差异 对于滋养层细胞的特定亚群，表达调节的实验已经证明 一般和亚型特异性调节条件，以及功能研究已经确定了定性和 两种异构体之间的定量差异。在本申请中，我们提出的研究将扩大我们的 这些强大的妊娠相关蛋白的知识。AIM 1旨在研究潜在的 单核吞噬细胞中的配体：受体相互作用，绘制表达图，并进行结合研究。 AIM 2的目标是建立G5和G6蛋白在细胞中差异表达的调节机制， 早期和晚期妊娠胎盘滋养层细胞亚群。在AIM 3中，目的是 研究G5和G6如何对单核吞噬细胞进行编程。我们将系统地比较 妊娠早期足月胎盘样本的结果和年龄匹配的正常胎盘样本的结果 问题妊娠的样本，即，先兆子痫和早产/分娩伴或不伴感染。 这项拟议中的研究与公共卫生的相关性建立在这样一个事实上，即至少有十分之一的夫妇 经历生育困难和高比例的怀孕失败，由于早产相关或不相关 感染了一个潜在的原因可能是G5/G6或其受体的合成或表达的异常。 LILRB1/LILRB2受体。我们期望在这里提供对设计至关重要的全新信息 的治疗策略，以解决HLA-G相关的病理妊娠。

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.