ERYTHROPOIETIN STRUCTURE-FUNCTION-RELATIONSHIPS - IDENTIFICATION OF FUNCTIONALLY IMPORTANT DOMAINS

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacement s at 51 conserved sites predicted to be on the surface of the molecule . Alanine replacements permitted preservation of alpha-helical structu re. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type an d mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell l ine, and the human UT7-EPO leukemia cell line. When Arg(14) On predict ed Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specif ic bioactivity. In a similar manner, the mutein Arg(103) --> Ala in He lix C was completely lacking in biological activity, whereas both Ser( 104) --> Ala and Leu(108) --> Ala had decreased bioactivity. In Helix D, the mutein Gly(15l) --> Ala had markedly decreased bioactivity, whe reas that of the adjacent Lys(152)--> Ala mutein was moderately impair ed. In contrast, Ala replacements at three nearby sites on Helix D (14 7, 146, and 143) resulted in muteins with increased bioactivity. In co nclusion, our mutagenesis experiments have identified functionally imp ortant domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.