Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure

J. P. Boissel, W. R. Lee, S. R. Presnell, F. E. Cohen, H. F. Bunn

Research output: Contribution to journalArticle

114 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)15983-15993
Number of pages11
JournalJournal of Biological Chemistry
Volume268
Issue number21
Publication statusPublished - 1993
Externally publishedYes

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Mutant Proteins
Erythropoietin
Bioactivity
Disulfides
Radioimmunoassay
Bioassay
Structural Models
Biological Assay
Serine
Tyrosine
Cells
Hormones
Cytokines
Cell Line

ASJC Scopus subject areas

  • Biochemistry

Cite this

Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure. / Boissel, J. P.; Lee, W. R.; Presnell, S. R.; Cohen, F. E.; Bunn, H. F.

In: Journal of Biological Chemistry, Vol. 268, No. 21, 1993, p. 15983-15993.

Research output: Contribution to journalArticle

Boissel, J. P. ; Lee, W. R. ; Presnell, S. R. ; Cohen, F. E. ; Bunn, H. F. / Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 21. pp. 15983-15993.
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