RED-CELL MEMBRANE REMODELING IN SICKLE-CELL-ANEMIA - SEQUESTRATION OFMEMBRANE-LIPIDS AND PROTEINS IN HEINZ BODIES

In red cells from patients with sickle cell anemia, hemoglobin S denat ures and forms Heinz bodies, Binding of Heinz bodies to the inner surf ace of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3; outer surface-bound autologous IgG an d, to some extent, the membrane proteins glycophorin and ankyrin, Loss of transbilayer lipid asymmetry is also found in certain populations of sickle red cells. The lateral distribution of sickle cell membrane lipids has not been examined, however. In this report, we examine by f luorescence microscopy the incorporation and distribution of the fluor escent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-p hosphatidylserine and NBD-phosphatidylcholine in sickle red cells, Bot h phospholipid analogues are observed to accumulate prominently at sit es of Heinz bodies. Accumulation at sites of Heinz bodies is also show n by 1,'1-dihexadecyl-3,3,3',3' -tetramethylindocarbocyanine perchlora te, a fluorescent Lipid analogue that readily crosses membranes, but n ot by fluorescein-phosphatidylethanolamine, an analogue that is locali zed to the outer leaflet of the membrane. Double labeling and confocal microscopy techniques show that NBD-lipids, band 3 protein, protein 4 .1, ankyrin, and spectrin are all sequestered within sickle red cells and colocalized at sites of Heinz bodies, We propose that Heinz bodies provide a hydrophobic surface on which sickle red cell membrane lipid s and proteins are sequestered.