Structure of Erythropoietin in African Clawed Frogs, Xenopus laevis, and Its Role in the Liver Erythropoiesis.

Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.