Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells.

250

115

A B S T R A C T In contrast to the wealth of infonnation concerning membrane phospholipid asymmetry in normal human erythrocytes, very little is known about membrane phospholipid organization in pathologic erythrocytes. Since the spectrin-actin lattice, which has been suggested to play an important role in stabilizing membrane phospholipid asymmetry, is abnormal in sickled erythrocytes, we determined the effects of sickling on membrane phospholipid organization. We used two enzymatic probes: bee venom phospholipase A2 and Staphylococcus aureus sphingomyelinase C, which do not penetrate the membrane and react only with phospholipids located in the outer leaflet of the bilayer. Our results suggest that the distribution of glycerophospholipids within the membrane of sickled cells is* different from that in nonsickled cells. Compared with the normal erythrocyte, the outer membrane leaflet of the deoxygenated, reversibly sickled cells (RSC) and irreversibly sickled cells (ISC) was enriched in phosphatidyl ethanolamine in addition to containing phosphatidyl serine. These changes were compensated for by a decrease in phosphatidyl choline in that layer. The distribution of sphingomyelin over the two halves of the bilayer was unaffected by sickling. In contrast to ISC, where the organization of phospholipids was abnormal under both oxy and deoxy conditions, reoxygenation of RSC almost completely restored the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane lipids in RSC which becomes permanent in ISC.

Section: Introductionmentioning

confidence: 99%

Abnormalities in membrane phospholipid organization in sickled erythrocytes.

Lubin¹,

Chiu²,

Bastacky³

et al. 1981

250

115

“…Irreversibly sickled cells (ISCs) are abnormally rigid (20) and manifest increased exposure of aminophospholipids in the outer leaflet of the RBC membrane (21)(22)(23)(24)(25). Membrane skeletons from ISCs retain the deformed sickle shape in the absence of hemoglobin and membrane lipid (26). Negative charges at the surface of sickle RBCs, presumably carried by glycophorins, may be abnormally clustered (27,28), although this finding is controversial (29).…”

Section: Introductionmentioning

confidence: 99%

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

Corbett¹,

Golan²

1993

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)

“…In the case of sickle cells, hypotheses relating the molecular defect in the hemoglobin to the altered properties of the membrane e.g., flexibility, Ca2+ permeability, lipid asymmetry, cytoskeletal structure, surface charge distribution, autologous antibody affinity, etc. (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), have generally attributed the membrane lesions either to the tendency of sickle hemoglobin (HbS)' to polymerize and distort the cell, or to its predisposition to denature and catalyze the production of oxidizing agents. In the latter hypothesis, the oxidizing agents are believed to react with membrane proteins and/or lipids in causing the defective membrane behavior (15).…”

Section: Introductionmentioning

confidence: 99%

Heinz bodies induce clustering of band 3, glycophorin, and ankyrin in sickle cell erythrocytes.

Waugh¹,

Willardson²,

Kannan³

et al. 1986

104

In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.