Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin.
Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.
Recny
M A
MA
Genetics Institute, Inc., Cambridge, Massachusetts 02140.
Scoble
H A
HA
Kim
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004274:DNA, Recombinant; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism* / Q000652:urine*; D006801:Humans; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000378:metabolism*; D010449:Peptide Mapping
D000596:Amino Acids; D004274:DNA, Recombinant; D010446:Peptide Fragments; C054371:erythropoietin, des-Arg(166)-; D004921:Erythropoietin