title: Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3.
abstract: Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.
authors.affiliation: Section of Internal Medicine, Department of Medicine,University of Verona, 37134 Verona, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Matte
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
authors.forename: Narla
authors.initials: N
authors.lastname: Mohandas
authors.forename: Xiuli
authors.initials: X
authors.lastname: An
authors.forename: Antonella
authors.initials: A
authors.lastname: Bugatti
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Brunati
authors.forename: Marco
authors.initials: M
authors.lastname: Rusnati
authors.forename: Elena
authors.initials: E
authors.lastname: Tibaldi
authors.forename: Angela
authors.initials: A
authors.lastname: Siciliano
authors.forename: Franco
authors.initials: F
authors.lastname: Turrini
authors.forename: Silverio
authors.initials: S
authors.lastname: Perrotta
authors.forename: Lucia
authors.initials: L
authors.lastname: De Franceschi
mesh_terms: D003593:Cytoplasm / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D054464:Peroxiredoxins / Q000378:metabolism*
chemical_list: C086065:PRDX2 protein, human; D054464:Peroxiredoxins