N-HOMOCYSTEINIYLATION ALTERS ERYTHROPOIETIN FUNCTIONS

MALTANERI R; NESSE A; SCHIAPPACASSE A; WETZLER D; CHAMORRO ME; VITTORI D

Mar del Plata

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

Sociedad Argentina de Investigación Clínica

Erythropoietin (Epo) is a cytokine known by its hematopoieticproperties. Despite the successful introduction of recombinant human Epo toovercome the anemia associated with different pathologies, a significant numberof patients fails to respond. High levels of homocysteine ?a risk factor forcardiovascular disease? have been linked with altered protein structure due toN-homocysteinylation of protein lysine residues by reaction with the highlyreactive homocysteinethiolactone (HTL). Therefore, it was interesting to studywhether the erythropoietic and antiapoptotic properties ofEpo changed afterN-homocysteinylation. Epo was incubated with HTL at 37 °C for 24 h (Epo:HTL1:2000 M) and residual HTL was removed by washing. Changes undergone by themolecule were evaluated by polyacrylamide gel electrophoresis, zone capillaryelectrophoresis, the Ellman reaction and fluorescence and circular dichroismmeasurements. Regarding erythropoietic action, Epo and Epo-HTL (8 ng/mL) werecompared in 48 h-growth of the Epo-dependent UT-7 cells capable of erythroiddifferentiation. Cell proliferation assay (trypan blue): Epo 45.2±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. Phosphatidylserinetranslocation was used to evaluate apoptosis (Flow cytometry, annexin V-positivecells): Epo 21.7 ± 2.6%; *EpoHTL 46.7 ± 1.5%; *P<0.05; n=4.The analysis of molecule alterations shows structural changes of Epo dueto HTL action. The results indicating reduced erythropoietic and antiapoptotic effectsof Epo-HTL on the UT-7 cell line, suggest a new possible form of erythropoietinresistance, especially important in patients with cardio-renal syndrome.