title: Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions.
abstract: Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT-PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT-PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.
authors.affiliation: Department of Environmental Sciences, University of Tuscia, Largo dell'Università snc, 01100 Viterbo, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Rinalducci
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: D'Amici
authors.forename: Barbara
authors.initials: B
authors.lastname: Blasi
authors.forename: Lello
authors.initials: L
authors.lastname: Zolla
mesh_terms: D001771:Blood Banks; D001793:Blood Preservation; D002374:Catalase / Q000097:blood*; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D003080:Cold Temperature; D057075:Enzyme Assays; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D018384:Oxidative Stress; D054464:Peroxiredoxins / Q000097:blood*; D011485:Protein Binding; D055503:Protein Multimerization; D013997:Time Factors
chemical_list: D054464:Peroxiredoxins; D002374:Catalase