Physiological roles of placental leucine aminopeptidase/oxytocinase and its clinical application using molecular biological technique.

胎盘亮氨酸氨基肽酶（P-LAP）与胱氨酸氨基肽酶相同，是胎盘和母体血清中主要的功能性催产素酶。本研究目的是利用分子生物学技术评价P-LAP的临床意义及应用。我们成功地建立了产生重组可溶性P-LAP的CHO细胞。重组P-LAP不仅能水解催产素和抗利尿激素，还能水解脑内运动啡肽A和脑啡肽。虽然需要进一步的体内研究，重组P-LAP可用于抑制分娩。P-LAP以膜结合形式和可溶性形式存在，这意味着可溶性形式通过特定的蛋白水解裂解释放。P-LAP分泌酶具有金属蛋白酶活性，可识别裂解位点的氨基酸序列进行攻击。我们已经分离出含有P-LAP基因5'上游区域的基因组克隆。我们证明了转录因子激活蛋白2 （AP-2）和Ikaros共同促进P-LAP在滋养细胞中的转录。白细胞介素- β (IL-1β)增加P-LAP活性和蛋白。半定量RT-PCR和Southern blotting显示，IL-1β也增加了P-LAP mRNA，而先前暴露于环己亚胺可以消除P-LAP mRNA。荧光素酶检测未发现任何可以解释il -1β诱导的P-LAP mRNA积累的调控区域。这些发现表明，长时间暴露于IL-1β可诱导胎盘中的P-LAP，可能是通过其他从头合成的蛋白质。透射免疫电镜显示P-LAP在合胞滋养细胞顶端微绒毛表面表达。在本研究中，我们获得了P-LAP缺陷小鼠和P-LAP过表达大鼠。目前，利用这些动物来评估自然分娩和催产素诱发的分娩变化的研究正在进行中。这一结果将阐明催产素酶在妊娠中的作用。

Placental leucine aminopeptidase (P-LAP), which is identical to cystine aminopeptidase, is the predominant and functional oxytocinase in the placenta and maternal serum. The aim of this study is to evaluate the clinical significance and application of P-LAP using molecular biological technique. We have succeeded in establishing the CHO cells producing recombinant soluble form of P-LAP. Recombinant P-LAP hydrolyses not only oxytocin and vasopressin, but also dynorphin A and enkephalin in the brain. Although further in vivo studies are required, recombinant P-LAP could be used to inhibit the labor. P-LAP exists in both membrane-bound and soluble forms, which means that the soluble form is released by a specific proteolytic cleavage. P-LAP secretase has a metalloprotease activity and recognizes the amino acid sequence of cleavage site to attack. We have isolated genomic clones containing the 5'-upstream region of the P-LAP gene. We demonstrated that transcription factors activating protein 2 (AP-2) and Ikaros cooperatively enhance P-LAP transcription in trophoblastic cells. Interleukin-lbeta (IL-1β) increased P-LAP activity and proteins. Semi-quantitative RT-PCR and Southern blotting showed that IL-1β also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1β-induced P-LAP mRNA accumulation. These findings indicated that prolonged exposure to IL-1β induces P-LAP in the placenta, possibly via other de novo protein synthesis. Transmission immunoelectron microscopy revealed that P-LAP was expressed on the surface of apical microvilli of syncytiotrophoblast cells. During this study, we could obtain both P-LAP-deficient mice and P-LAP overexpressing rats. Now the studies employing these animals to evaluate the changes of the spontaneous and oxytocin-induced labor are on the way. The results would elucidate the roles of oxytocinase during pregnancy.