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 the present study, using a variety of experimental approaches including crosslinking studies, fluorescence and dichroic measurements, surface-plasmon-resonance analysis, and proteolytic digestion assays, we document that the anti-oxidant 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 interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine (PHZ) of RBCs prevented membrane association of PRDX2 implying over lapping 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 acid 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.

Membrane association of peroxiredoxin-2 in red cells is mediated by n-terminal cytoplasmic domain of band 3.

PERROTTA, Silverio;
2013

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 the present study, using a variety of experimental approaches including crosslinking studies, fluorescence and dichroic measurements, surface-plasmon-resonance analysis, and proteolytic digestion assays, we document that the anti-oxidant 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 interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine (PHZ) of RBCs prevented membrane association of PRDX2 implying over lapping 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 acid 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11591/191508
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