Nagasawa Kazumichi, Meguro Mizue, Sato Kei, Tanizaki Yuta, Nogawa-Kosaka Nami, Kato Takashi
Integrative Bioscience and Biomedical Engineering, Graduate School of Advanced Science and Engineering, Waseda University, Center for Advanced Biomedical Science, TWIns building, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
Integrative Bioscience and Biomedical Engineering, Graduate School of Advanced Science and Engineering, Waseda University, Center for Advanced Biomedical Science, TWIns building, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Department of Biology, School of Education, Waseda University, Center for Advanced Biomedical Science, TWIns building, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
PLoS One. 2015 Apr 21;10(4):e0124676. doi: 10.1371/journal.pone.0124676. eCollection 2015.
Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1-3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.
促红细胞生成素（EPO）是红细胞生成的主要调节因子，是一种在人类和其他几种哺乳动物中发现的高度糖基化蛋白。有趣的是，我们之前发现非洲爪蟾（xlEPO）中的EPO没有N-糖基化位点，并且尽管与人类EPO（huEPO）的同源性较低，但仍能与人EPO受体发生交叉反应。在本研究中，我们在与huEPO同源的位置将N-糖基化位点引入野生型xlEPO，并测试糖基化突变体是否保留其生物活性。在COS-1细胞中表达了7种xlEPO突变体，它们在第24、38和/或83位含有1-3个额外的N-连接碳水化合物。与野生型相比，这些突变体表现出较低的分泌效率、较高的亲水性和较强的酸性。所有突变体均以剂量依赖性方式刺激两种细胞系的增殖，即表达xlEPO受体的xlEPOR-FDC/P2细胞和表达huEPO受体的UT-7/EPO细胞。因此，这些突变体保留了它们的体外生物活性。添加N-连接碳水化合物会降低对xlEPOR-FDC/P2增殖的最大影响，但对UT-7/EPO增殖的影响不变，这表明这些突变体对xlEPO受体起部分激动剂作用，对huEPO受体起近乎完全激动剂作用。因此，非洲爪蟾中的EPO-EPOR结合位点位于人工引入的三个N-糖基化位点的远端区域，这表明尽管EPO和EPOR的一级结构相似性较低，但在人类和非洲爪蟾之间发挥生物活性的关键构象是保守的。
