Structural role of amino acids 99–110 in recombinant human erythropoietin

Authors

  • Yijuang CHERN,

    1. Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Taeowan CHUNG,

    1. Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Arthur J. SYTKOWSKI

    Corresponding author
    1. Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, MA, USA
      Correspondence to A. J. Sytkowski, Laboratory for Cell and Molecular Biology, New England Deaconess Hospital, 185 Pilgrim Road, BURL Bldg. 5th Floor. Boston, MA02215, USA
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Correspondence to A. J. Sytkowski, Laboratory for Cell and Molecular Biology, New England Deaconess Hospital, 185 Pilgrim Road, BURL Bldg. 5th Floor. Boston, MA02215, USA

Abstract

Erythropoietin is the prime regulator of red blood cell production. Previous studies demonstrated that antipeptide antibodies to amino acids 99–119 and 111–129 bind to two non-overlapping domains and inhibit the hormone's action (Sytkowski, A. J. & Donahue, K. A. (1987) J. Biol. Chem. 262, 1161–1165). Oligonucleotide-directed mutagenesis now shows that amino acids 99–110 (domain 1) but not 119–129 (domain 2) are important to erythropoietin's structure and function. Mutagenesis of wild-type human erythropoietin cDNA was used to produce a series of mutant proteins with sequential deletion of three adjacent amino acids and insertion of the sequence Glu-Phe across the two domains. Transient expression in COS-7 cells revealed 2.0-kb transcripts encoded by all of the cDNAs. Domain 2 mutants exhibited specific biological activities similar to that of the wild type. In contrast, domain 1 mutants were not secreted. In vitro transcription and translation of the domain 1, domain 2 and wild-type cDNAs resulted in the isolation of 23.5-kDa and 32-kDa proteins in the absence or presence of pancreatic microsomes, respectively, consistent with efficient translation of all of the mutants and equivalent post-translational processing of each protein. The data suggest that mutation within domain 1 results in the intracellular biosynthesis of erythropoietins with altered structure, rendering them subject to rapid degradation. The bioassay of erythropoietins synthesized entirely in vitro demonstrated that domain 1 mutants were inactive, whereas both wild type and domain 2 mutant hormones exhibited biologic activity. The results are consistent with a critical role for amino acids 99–110 in the structure of human erythropoietin.

Abbreviations
Epo

erythropoietin cDNA insert

wt

wild type