Band 3 and glycophorin are progressively aggregated in density-fractionated sickle and normal red blood cells. Evidence from rotational and lateral mobility studies.

Abstract

Band 3 aggregation in the plane of the red blood cell (RBC) membrane is postulated to be important in the pathophysiology of hemolysis of dense sickle and normal RBCs. We used the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3, glycophorins, and phospholipid analogues in membranes of density-separated intact RBCs from seven patients with sickle cell disease and eight normal controls. The fractions of laterally mobile band 3 and glycophorin decreased progressively as sickle RBC density increased. Normal RBCs also showed a progressive decrease in band 3 fractional mobility with increasing buoyant density. Rapidly rotating, slowly rotating, and rotationally immobile forms of band 3 were observed in both sickle and normal RBC membranes. The fraction of rapidly rotating band 3 progressively decreased and the fraction of rotationally immobile band 3 progressively increased with increasing sickle RBC density. Changes in the fraction of rotationally immobile band 3 were not reversible upon hypotonic swelling of dense sickle RBCs, and normal RBCs osmotically shrunken in sucrose buffers failed to manifest band 3 immobilization at median cell hemoglobin concentration values characteristic of dense sickle RBCs. We conclude that dense sickle and normal RBCs acquire irreversible membrane abnormalities that cause transmembrane protein immobilization and band 3 aggregation. Band 3 aggregates could serve as cell surface sites of autologous antibody binding and thereby lead to removal of dense sickle and normal (senescent)

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

“…Lateral and rotational mobility of fluorescently labeled band 3 protein, red cell glycophorins, and membrane lipids were measured in intact washed red cells, as described (31). Briefly, band 3 protein was labeled with E5M, glycophorins were labeled with fluorescein thiosemicarbazide (Molecular Probes), and a fluorescent lipid analog, fluorescein phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL), was introduced into the intact red cell membrane as described (31 ). Lateral mobility of the labeled membrane components was measured by fluorescence photobleaching recovery (31 ) and rotational mobility of band 3 was measured by polarized fluorescence depletion (31,32).…”

“…Previous studies of lateral and rotational mobility of band 3 protein in the normal red cell membrane have revealed the presence of several distinct band 3 fractions. In terms of lateral mobility, two principal fractions have been identified: (a) an immobile fraction that most likely represents ankyrin-linked band 3 tetramers and higher order oligomers; and (b) a mobile fraction that most likely represents band 3 species that are not directly associated with the membrane skeleton and can, therefore, migrate laterally over m-scale distances in the plane ofthe lipid bilayer (31,40). Rotational mobility studies have previously identiband3dimers Figure 6.…”

“…Rotational mobility studies have previously identiband3dimers Figure 6. Analysis of ankyrin/band 3 com- fied three band 3 populations: (a) a rotationally immobile fraction of band 3 molecules thought to comprise proteins bound directly to ankyrin; (b) a slowly rotating population believed to represent band 3 oligomers that are either bound indirectly to ankyrin or present as larger aggregates in the membrane; and (c) a rapidly rotating fraction consisting of band 3 species (primary dimeric and tetrameric) that rotate freely in the lipid bilayer (31,41 ). Thus, studies of band 3 lateral and rotational mobility in our HS kindred could provide an assessment of band 3 self-association and ofthe interaction ofband 3 with the skeleton in the intact membrane, and thereby complement our results from the HPLC studies ofthe oligomeric species ofband 3 described above.…”

C3b deposition on human erythrocytes induces the formation of a membrane skeleton–linked protein complex

“…rbc were washed 3 times with HBS, resuspended at 10% hematocrit in HBS with 10 mM glucose, and kept at 4°C until use. Alternatively, rbc were labeled with fluorescein thiosemicarbazide to derivatize cell-surface GPA (53). rbc (1 × 10 8 ) were washed twice in high-potassium PBS ([KPBS] 140 mM KCl, 15 mM Na2HPO4, pH 7.4), incubated with 1 mM NaIO4 in 200 μl of KPBS for 15 minutes at 4°C, washed twice in 1 ml of KPBS with 0.1 M glycerol and once in KPBS, resuspended in 200 μl of KPBS, and mixed with 200 μl of 0.5 mg/ml fluorescein thiosemicarbazide in KPBS.…”

Differential control of band 3 lateral and rotational mobility in intact red cells.

“…The mobile fraction of band 3 diffuses laterally at a rate of 1-2 X 10-11 cm2 s-' (17- 19). In contrast, RBC phospholipid analogues have fractional mobilities of 90-100% and translational diffusion coefficients of 1-5 x 10-9 cm2 S-' (17, 20).…”