Structural and biological properties of erythropoietin in Xenopus laevis
Received 4 December 2009; received in revised form 4 February 2010; accepted 22 February 2010. published online 02 March 2010.
Objective
Erythropoietin (EPO) and its receptor (EPOR) are key regulators of red blood cell production in mammals and fish. We aimed to investigate the structural and functional conservation of the EPO-EPOR system in amphibian erythropoiesis, using Xenopus laevis as a model.
Materials and Methods
X. laevis epo (xlepo) complementary DNA was identified by referring to the Xenopus tropicalis genome database. Biological activity of recombinant xlEPO expressed in COS-1 cells was evaluated using xlEPOR-expressing murine FDC/P2 cells and human EPO-dependent UT-7/EPO cells. Expression of xlepo messenger RNA in adult X. laevis tissues in the normal state and under the condition of phenylhydrazine-induced anemia was evaluated by real-time reverse transcription polymerase chain reaction.
Results
In the encoded protein, the positions of four cysteine residues were conserved; however, xlEPO had only 38% identity with human EPO. N-glycosylation sites were absent. Recombinant xlEPO induced proliferation of cell lines expressing xlEPOR and UT-7/EPO, confirming biological activity and cross-species reactivity. Despite little primary amino acid sequence similarity, the evolutionary highly conserved sequence NFLRGK was identified in the EPOR-binding site 1 region as in the human EPO protein. Strong expression of xlepo messenger RNA was detected in the lung and liver, especially in fractionated hepatocytes. No marked increase in xlepo expression was seen in the lung and liver of phenylhydrazine-induced anemic X. laevis.
Conclusion
We confirmed that xlEPO is the ligand to the previously reported xlEPOR in X. laevis. xlEPO shares structural and functional similarities and differences with mammalian counterparts, and regulation of xlepo expression and its influence on the erythropoietic system appears to be unique.
aGraduate School of Advanced Science and Engineering, Center for Advanced Life and Medical Science, Waseda University, Tokyo, Japan
cDepartment of Hematology, Faculty of Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan
dDepartment of Biology, School of Education, Waseda University, Tokyo, Japan
Offprint requests to: Takashi Kato, D.Sc., Faculty of Education and Integrated Arts and Sciences, Center for Advanced Life and Medical Science, TWIns building, Waseda University, 2-2 Wakamatsu-cho Shinjuku Tokyo 162-8480, Japan
∗ Dr. Komatsu's current address: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
ABSTRACT