Identification and functional analysis of two unknown PHGPx in embryogenesis

磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）是一种独特的能直接还原磷脂氢过氧化物的抗氧化酶。小鼠PHGPx基因的破坏导致胚胎死亡率为8.5%。然而，关于PHGPx在胚胎发生中的作用的分析仍然存在。为了明确PHGPx在胚胎发生中的作用，本研究采用体外培养系统和转基因拯救法检测了PHGPx蛋白和mRNA在胚胎发生中的表达，并对其功能进行了分析。我们发现在7.5dpc-9.5dpc胚胎中有新的18kDa的PHGPx表达。该PHGPx通过二硫形成与其他蛋白结合。通过5′race分析，在lb外显子中发现了新的转录星形位点和新的翻译起始位点。体外培养系统中，PHGPx KO胚胎死亡，不能形成ICM，但转染非线粒体PHGPx挽救了KO胚胎的致死性。我们开发了新的策略来分析PHGPx在胚胎发生中的作用。正常PHGPx基因的转基因恢复了KO小鼠的致死率，而PHGPx基因的所有突变体均未恢复KO小鼠的致死率。在胎盘中，我们发现20kDa的PHGPx与10kDa的其他蛋白和二聚体形成相关。

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide. Disruption of PHGPx gene in mice resulted in the embryonic lethality at 8.5 dpc. However, the analysis of role of PHGPx in embryogenesis has remained.In this study, to clarify the role of PHGPx during embryogenesis, we examined about the expression of PHGPx protein, mRNA in embryogenesis and the functional analysis using in vitro culture system and transgenic rescue method.We found that new 18kDa PHGPx was expressed in 7.5dpc-9.5dpc embryos.This PHGPx was associated with other protein via disulfide formation.From 5'RACE analysis, new transcriptional star site in lb exons was found and new translational start site. In vitro embryo culture system, PHGPx KO embryo died and could not formation of ICM, however transfection of non-mitochondrial PHGPx rescued lethality of KO embryo. We developed new strategy for analysis of role of PHGPx during embryogenesis using transgenic rescue. Trans gene of normal PHGPx gene rescued lethality of KO mice, but all mutant of PHGPx gene did not rescued lethality of KO mice. In placenta we found that 20kDa PHGPx was associated with 10kDa other protein and dimmer formation.