title: Structural and biological properties of erythropoietin in Xenopus laevis.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Graduate School of Advanced Science and Engineering, Center for Advanced Life and Medical Science, Waseda University, Tokyo, Japan.
authors.forename: Nami
authors.initials: N
authors.lastname: Nogawa-Kosaka
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hirose
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Kosaka
authors.forename: Youichi
authors.initials: Y
authors.lastname: Aizawa
authors.forename: Kazumichi
authors.initials: K
authors.lastname: Nagasawa
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Uehara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyazaki
authors.forename: Norio
authors.initials: N
authors.lastname: Komatsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000743:Anemia, Hemolytic / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017849:Animals, Inbred Strains; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004920:Erythropoiesis; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010659:Phenylhydrazines / Q000633:toxicity; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D010659:Phenylhydrazines; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C030299:phenylhydrazine; D004921:Erythropoietin