title: The influence of artificially introduced N-glycosylation sites on the in vitro activity of Xenopus laevis erythropoietin.
abstract: 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.
authors.affiliation: 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.
authors.forename: Kazumichi
authors.initials: K
authors.lastname: Nagasawa
authors.affiliation: 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.
authors.forename: Mizue
authors.initials: M
authors.lastname: Meguro
authors.affiliation: 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.
authors.forename: Kei
authors.initials: K
authors.lastname: Sato
authors.affiliation: 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.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Tanizaki
authors.affiliation: 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.
authors.forename: Nami
authors.initials: N
authors.lastname: Nogawa-Kosaka
authors.affiliation: 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.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002236:Carbohydrate Conformation; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D014162:Transfection; D019076:Transgenes; D014982:Xenopus laevis
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin