Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin.
Physicochemical and biological properties of recombinant human erythropoietin (rhEPO) were compared with human urinary erythropoietin (uEPO). uEPO and rhEPO were purified to apparent homogeneity from the urine of patients with aplastic anemia and from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human EPO, respectively. The microheterogeneous nature of both factors, observed on isoelectric focusing, is derived from the difference of the number of terminal sialic acid residues bound to the carbohydrate chains of the EPO molecule. The primary structure of rhEPO, consisting of 165 amino acid residues, was determined, and the C-terminal arginine predicted from the cDNA sequence was confirmed to be missing, as described previously (Recny et al. (1987) J. Biol. Chem. 262, 17156). Three N-glycosylation and one O-glycosylation sites of both factors were determined as Asn24, Asn38, and Asn83 and Ser126, respectively. Two disulfide linkages are located between Cys7 and Cys161, and between Cys29 and Cys33, in both EPOs. Hematogenic potencies of rhEPO and uEPO compared in normal and in partially nephrectomized rats were approximately the same. Both factors also stimulated the colony formation of CFU-E, BFU-E, and CFU-Meg in a dose-dependent manner. From these results, it is concluded that rhEPO produced in CHO cells transfected with cDNA clone for human EPO is indistinguishable from uEPO physicochemically and biologically, and is valuable for further research and for clinical use.
Imai
N
N
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka.
Kawamura
A
A
Higuchi
M
M
Oh-eda
M
M
Orita
T
T
Kawaguchi
T
T
Ochi
N
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000741:Anemia, Aplastic / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000032:analysis* / Q000494:pharmacology / Q000652:urine; D006410:Hematopoiesis / Q000187:drug effects; D006801:Humans; D007501:Iron / Q000378:metabolism; D007504:Iron Radioisotopes; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D007504:Iron Radioisotopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D004921:Erythropoietin; D007501:Iron