Modification of erythropoietin structure by N‐homocysteinylation affects its antiapoptotic and proliferative functions

Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N‐homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N‐homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL‐EPO. A decreased net positive charge of HTL‐EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far‐UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α‐helix to β‐sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive β‐sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL‐EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand‐receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio‐renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N‐homocysteinylation. The N‐homocysteinylation of erythropoietin alters its erythropoietic and antiapoptotic activities due to the highly reactive homocysteine thiolactone (HTL) reacting with erythropoietin's lysine residues. This functional alteration might be caused by structural changes to erythropoietin that switch the protein's secondary structure from an α‐helix to a β‐sheet configuration; this configuration forms a repetitive β‐sheet, consistent with the development of soluble oligomers.