Alanine scanning mutagenesis of human erythropoietin identifies four amino acids which are critical for biological activity.
Erythropoietin regulates the growth and proliferation of red blood cell progenitors. We demonstrated previously the important structural and functional roles of the amino acid region 99-110 in the biological activity of the hormone. [Chern, Y., Chung, T. & Sytkowski, A. J. (1991) Eur. J. Biochem. 202, 225-229]. We have now performed alanine scanning mutagenesis to identify which specific residues in this region are essential for function. Four substitutions reduced the biological activity of erythropoietin. Alanine substitution of Ser104, Leu105 and Leu108 reduced activity to 16, 44, and 37% that of the wild-type protein, respectively. Most significantly, substitution of Arg103 with Ala reduced activity to undetectable levels, indicating a > 230-fold reduction in specific activity. Immunochemical analyses using anti-peptide monoclonal antibodies demonstrated that this substitution does not cause major changes in the conformation of the protein or large, localized distortions within the amino acid region 99-129 of the molecule. Heat-denaturation kinetics for the arginine mutant and wild-type erythropoietin are virtually identical, further indicating the structural similarity between these two molecules. Based upon these findings, we propose that Arg103 plays an essential role in the biological activity of erythropoietin, presumably by interacting directly with the erythropoietin receptor.
Grodberg
J
J
New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston 02215.
Davis
K L
KL
Sykowski
A J
AJ
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007120:Immunochemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis
D000596:Amino Acids; D018076:DNA, Complementary; D004921:Erythropoietin; D000409:Alanine