Conformational changes in membrane protein Band 3 associated with erythrocyte aging

PLATERO E; ENSINCK A; RUZZINI M; RACCA L; COTORRUELO C; RACCA A; RUCCI A

Amsterdam

Congreso; 23rd Congress of the International Society of Blood Transfusion; 2013

International Society of Blood Transfusion

Background: Band 3 (AE1) belongs to a ubiquitous family of anion exchange protein that are widely distributed in the membranes of diverse cell types. AE1 is the major membrane protein of red blood cells (RBCs), containing 911 amino acids and a membrane-spanning domain, traversing the bilayer 14 times, in ¦Á-helical segments highly conserved. Physicochemical changes of band 3 during erythrocyte aging are probably part of the mechanism leading to the generation of a senescent cell antigen (SCA). It serves as a specific signal for the clearance of senescent (Se) RBCs by inducing the binding of autologous IgG and phagocytosis. 4,4-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) is a competitive inhibitor of Cl¡¥/HCO3¡¥ exchange by joining Lys 539 or Lys 542 of the 5th ¦Á-helical segment of band 3. This compound can be used to study conformational changes of this protein during erythrocyte aging. Aims: The aim of this study was to evaluate the conformational changes of the band 3 protein, analyzing the spectra of adduct DIDS-band 3, in young (Y) RBC and SeRBC populations. Methods: We studied peripheral blood samples of volunteer donors with negative serology (n=8). SeRBCs and YRBCs were obtained subjecting the samples to preformed Percoll density gradients. The separation efficiency was evaluated by the mean corpuscular volume. Samples were incubated for 45 min at 37¡ãC in 25 mM DIDS and then lysed with 5 mM phosphate buffer pH 8. Subsequently, the pellets obtained were washed and dissolved in a 1% octaethylene glycol monododecyl ether solution. Fluorescence measurement was performed on a Varian Cary Eclipse spectrophotometer. The excitation spectra were performed between 300 and 450 nm (¦Ëem = 460 nm). The emission spectra were collected between 360 and 600 nm (¦Ëexc = 350 nm). Both spectra were performed for DIDS free (DF), aduct DIDS-band 3 in SeRBCs (DS) and aduct DIDS-band 3 in YRBCs (DY). Comparisons of the values of different variables obtained in SeRBC and YRBC populations were performed by means of the t-test for paired samples. Results: The results are shown in the table. Statistical analysis between the emission peaks in different populations showed a greater shift in YRBCs (p<0.05), whereas the shift of excitation peaks were greater in SeRBCs (p<0.05). Conclusions: The results show significant changes in the spectra of aduct DIDS-band 3, suggesting alterations in the tertiary structure of AE1 associated with aging. These modifications could be involved in the generation of the SCA, a specific signal for the clearance of SeRBCs.