Immunochemical studies of human erythropoietin using site-specific anti-peptide antibodies. Identification of a functional domain.
Anti-peptide antibodies that bind to the amino terminus of human erythropoietin (residues 1-26) do not inhibit the hormone's biological activity, indicating that this region of the protein does not play a role in receptor recognition (Sytkowski, A. J., and Fisher, J. W. (1985) J. Biol Chem. 260, 14727-14731). We have now identified six other regions of the primary sequence that are relatively hydrophilic and, therefore, have a higher probability of being accessible to such antibody probes. Antibodies raised against synthetic peptides homologous to five of these regions, corresponding to residues 40-59, 80-99, 99-118, 111-129, and 131-150 recognize erythropoietin, confirming the prediction based upon relative hydrophilicity. Antibodies to a carboxyl terminal peptide 147-166 failed to bind the hormone, presumably due to steric hindrance imposed by a disulfide bond between Cys161 and one of the other cysteinyl residues. The antibodies were affinity purified on the relevant immobilized peptide and their capacity to inhibit (neutralize) erythropoietin's activity was assessed. Only anti-peptide 99-118 and anti-peptide 111-129 antibodies inhibited erythropoietin. This effect was reversed by excess peptide, demonstrating that the neutralizing action of the antibody was due to its antigen-specific binding. The results strongly suggest that the portion of erythropoietin's amino acid sequence represented by these peptides plays a functional role in the hormone's action, most probably by forming part of the receptor-binding domain.
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D004921:Erythropoietin / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D017467:Receptors, Erythropoietin
D000906:Antibodies; D000941:Antigens; D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin