Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure.
On the basis of its primary sequence and the location of its disulfide bonds, we propose a structural model of the erythropoietic hormone erythropoietin (Epo) which predicts a four alpha-helical bundle motif, in common with other cytokines. In order to test this model, site-directed mutants were prepared by high level transient expression in Cos7 cells and analyzed by a radioimmuno assay and by bioassays utilizing mouse and human Epo-dependent cell lines. Deletions of 5 to 8 residues within predicted alpha-helices resulted in the failure of export of the mutant protein from the cell. In contrast, deletions at the NH2 terminus (delta 2-5), the COOH terminus (delta 163-166), or in predicted interhelical loops (AB: delta 32-36, delta 53-57; BC: delta 78-82; CD: delta 111-119) resulted in the export of immunologically detectable Epo muteins that were biologically active. The mutein delta 48-52 could be readily detected by radioimmunoassay but had markedly decreased biological activity. However, replacement of each of these deleted residues by serine resulted in Epo muteins with full biological activity. Replacement of Cys29 and Cys33 by tyrosine residues also resulted in the export of fully active Epo. Therefore, this small disulfide loop is not critical to Epo's stability or function. The properties of the muteins that we tested are consistent with our proposed model of tertiary structure.
Boissel
J P
JP
Hematology/Oncology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Lee
W R
WR
Presnell
S R
SR
Cohen
F E
FE
Bunn
H F
HF
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D004220:Disulfides; D004921:Erythropoietin