N-homocysteiniylation alters erythropoietin functions.

MALTANERI ROMINA EUGENIA; CHAMORRO MARÍA EUGENIA; NESSE, ALCIRA; VITTORI DANIELA; SCHIAPPACASSE AGUSTINA; WETZER DIANA

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2016

Erythropoietin (Epo)is a cytokine known by its hematopoietic properties. Despite the successfulintroduction of recombinant human Epo to overcome the anemia associated withdifferent pathologies, a significant number of patients fails to respond. Highlevels of homocysteine ?a risk factor for cardiovascular disease? have beenlinked with altered protein structure due to N-homocysteinylation of proteinlysine residues by reaction with the highly reactive homocysteinethiolactone(HTL). Therefore, it was interesting to study whether theerythropoietic andantiapoptoticproperties ofEpochanged after N-homocysteinylation. Epo wasincubated with HTL at 37 °C for 24 h (Epo:HTL 1:2000 M) and residual HTL wasremoved by washing. Changes undergone bythe molecule were evaluated bypolyacrylamide gel electrophoresis, zone capillary electrophoresis, the Ellmanreaction and fluorescence and circular dichroism measurements. Regardingerythropoieticaction,Epo and Epo-HTL (8 ng/mL) were compared in 48 h-growth of theEpo-dependentUT-7 cells capable oferythroid differentiation. Cell proliferationassay (trypan blue): Epo45.2±8.8 x104cell/mL; *EpoHTL 29.6±2.7x104cell/ml;*P<0.05(*P<0.05), n=8; MTT assay: Epo 0.892 ± 0.071 O.D.; **EpoHTL 0.502± 0.061 O.D.; **P<0.01; n=8.Phosphatidylserine translocation was used toevaluate apoptosis (Flow cytometry, annexin V-positive cells): Epo 21.7 ± 2.6%;*EpoHTL 46.7 ± 1.5%; *P<0.05; n=4.The analysis of moleculealterations shows structural changes ofEpodue to HTL action. The results indicatingreduced erythropoietic and antiapoptotic effects of Epo-HTL on the UT-7 cellline, suggest a new possible form of erythropoietin resistance, speciallyimportant in patients with cardio-renal syndrome.