Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage.
Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.
Rybicki
A C
AC
Division of Hematology, Albert Einstein College of Medicine-Montefiore Medical Center, Bronx, NY 10467, USA.
Schwartz
R S
RS
Hustedt
E J
EJ
Cobb
C E
CE
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification; D016880:Anisotropy; D001798:Blood Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005454:Fluorescence Polarization; D005960:Glucosides; D006801:Humans; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010641:Phenotype; D012399:Rotation; D012997:Solvents
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D005960:Glucosides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D012997:Solvents; C044791:erythrocyte membrane band 4.2 protein; C018619:octyl-beta-D-glucoside