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

作者：WEN DY; BOISSEL JP; SHOWERS M; RUCH BC; BUNN HF

来源：Journal of Biological Chemistry, 1994, 269(36): 22839-22846.

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacements at 51 conserved sites predicted to be on the surface of the molecule. Alanine replacements permitted preservation of alpha-helical structure. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type and mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell line, and the human UT7-EPO leukemia cell line. When Arg(14) On predicted Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specific bioactivity. In a similar manner, the mutein Arg(103) --> Ala in Helix 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, whereas that of the adjacent Lys(152)--> Ala mutein was moderately impaired. In contrast, Ala replacements at three nearby sites on Helix D (147, 146, and 143) resulted in muteins with increased bioactivity. In conclusion, our mutagenesis experiments have identified functionally important domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.

• 出版日期1994-9-9