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 α-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 radioimmunoassay and by bioassays utilizing mouse and human Epo-dependent cell lines. Deletions of 5 to 8 residues within predicted α- helices resulted in the failure of export of the mutant protein from the cell. In contrast, deletions at the NH 2 terminus (Δ2-5), the COOH terminus (Δ163-166), or in predicted interhelical loops (AB: Δ32-36, Δ53-57; BC: Δ78-82; CD: Δ111-119) resulted in the export of immunologically detectable Epo muteins that were biologically active. The mutein Δ48-52 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 Cys 29 and Cys 33 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.
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1993|
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