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.
Highlights
Erythropoietin (EPO) is a hematopoietic cytokine that regulates the rate of red blood cell production [1, 2]
We introduced N-glycosylation consensus sequences into the X. laevis EPO (xlEPO) at positions homologous to those of human EPO (huEPO) (i.e., Asn24, Asn38, and Asn83) using in vitro mutagenesis (Fig 1A)
Non-glycosylated Asn83 was observed in xlEPO-3, xlEPO-13, xlEPO-23, and xlEPO-123, which indicates that carbohydrate additions were efficient at Asn24 and Asn38, but inefficient at Asn83
Summary
Erythropoietin (EPO) is a hematopoietic cytokine that regulates the rate of red blood cell production [1, 2]. We found that xlEPO stimulates the proliferation of the cells that express human EPOR (huEPOR), even though the amino acid sequence of xlEPO is only 38% identical to that of huEPO [21] This led us to expect that the functional region of EPO, which is responsible for in vitro activity, is conserved between X. laevis and humans. We tested this hypothesis by examining the activity of xlEPO muteins containing artificial N-glycosylation sites at positions homologous to those in huEPO (i.e., Asn, Asn, and Asn83) Those xlEPO muteins retained in vitro activity, demonstrating that the fundamental conformations of EPO-EPOR binding are conserved among humans and X. laevis; despite the low similarity in the primary sequences. For reporting the comparative analysis of EPO in this study, we named the X. laevis erythropoietin protein “xlEPO” instead of xlepo
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