Sun Irene Y C, Overgaard Michael T, Oxvig Claus, Giudice Linda C
Department of Gynecology and Obstetrics, Center for Research on Women's Health and Reproductive Medicine, Stanford University Medical Center, Stanford, CA 94305-5317, USA.
J Clin Endocrinol Metab. 2002 Nov;87(11):5235-40. doi: 10.1210/jc.2002-020561.
Pregnancy-associated plasma protein-A (PAPP-A) is a product of the placenta and decidua and is secreted into the maternal circulation during human pregnancy. It recently has been identified as an IGF binding protein (IGFBP)-4 protease. Presumed functions at the maternal-fetal interface are to proteolyze IGFBP-4 and thus increase IGF bioavailability locally in the placenta, to promote IGF-II-mediated trophoblast invasion into the maternal decidua, and to modulate IGF regulation of steroidogenesis and glucose and amino acid transport in the villous. Herein, we have investigated the possibility that IGFBP-4 proteolysis may occur on the trophoblast cell membrane, presumably to increase local bioavailable IGF for interactions with cognate IGF membrane receptors. Human trophoblasts were cultured, trophoblast plasma membranes were isolated and solubilized, and IGFBP-4 protease activity and PAPP-A immunoreactivity in the solubilized plasma membrane fraction were investigated. IGFBP-4 protease activity was detected in solubilized human trophoblast membranes, resulting in cleavage of recombinant human IGFBP-4 into 18- and 14-kDa fragments, detected by Western immunoblot analysis. This protease activity was dependent on the presence of IGF-II, and its metal ion dependence was demonstrated by inhibition of the protease by the metal chelators, EDTA and EGTA. The presence of PAPP-A in solubilized human trophoblast membranes was demonstrated by Western immunoblotting. Trophoblast membrane PAPP-A had a relative molecular weight of approximately 200 kDa and comigrated on (reducing) SDS-PAGE with recombinant human PAPP-A and PAPP-A secreted into media conditioned by cultured human trophoblasts. IGFBP-4 protease activity was not detected after immunodepletion of PAPP-A from the trophoblast membrane fraction with PAPP-A polyclonal antibodies, suggesting the identity of the membrane-derived IGFBP-4 protease as PAPP-A. Immunocytochemistry revealed PAPP-A on the cell membrane and in the cytoplasm of human trophoblasts in culture. Together, these data demonstrate the presence of an IGF-II- and metal-dependent IGFBP-4 protease activity in human trophoblast plasma membranes, identified as PAPP-A, which is well situated to proteolyze IGFBP-4 at the maternal-placental interface to facilitate IGF action at the villous surface and/or the invading extravillous cytotrophoblast.
妊娠相关血浆蛋白-A（PAPP-A）是胎盘和蜕膜的产物，在人类妊娠期间分泌到母体循环中。最近它被鉴定为一种胰岛素样生长因子结合蛋白（IGFBP）-4蛋白酶。在母胎界面推测的功能是蛋白水解IGFBP-4，从而在胎盘中局部增加IGF的生物利用度，促进IGF-II介导的滋养层细胞侵入母体蜕膜，并调节绒毛中类固醇生成以及葡萄糖和氨基酸转运的IGF调节。在此，我们研究了IGFBP-4蛋白水解可能发生在滋养层细胞膜上的可能性，推测这是为了增加局部可利用的IGF以与同源IGF膜受体相互作用。培养人滋养层细胞，分离并溶解滋养层细胞膜，研究溶解的细胞膜部分中的IGFBP-4蛋白酶活性和PAPP-A免疫反应性。在溶解的人滋养层细胞膜中检测到IGFBP-4蛋白酶活性，通过Western免疫印迹分析检测到重组人IGFBP-4被切割成18 kDa和14 kDa的片段。这种蛋白酶活性依赖于IGF-II的存在，并且其金属离子依赖性通过金属螯合剂EDTA和EGTA对蛋白酶的抑制得以证明。通过Western免疫印迹证明了溶解的人滋养层细胞膜中存在PAPP-A。滋养层细胞膜PAPP-A的相对分子量约为200 kDa，在（还原）SDS-PAGE上与重组人PAPP-A以及分泌到培养的人滋养层细胞条件培养基中的PAPP-A共迁移。用PAPP-A多克隆抗体从滋养层细胞膜部分免疫去除PAPP-A后未检测到IGFBP-4蛋白酶活性，表明膜来源的IGFBP-4蛋白酶与PAPP-A相同。免疫细胞化学显示培养的人滋养层细胞的细胞膜和细胞质中有PAPP-A。总之，这些数据证明人滋养层细胞膜中存在一种依赖IGF-II和金属的IGFBP-4蛋白酶活性，鉴定为PAPP-A，它在母胎界面处能够很好地蛋白水解IGFBP-4，以促进IGF在绒毛表面和/或侵入的绒毛外细胞滋养层中的作用。
