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. 1996 Jan 1;97(1):29–36. doi: 10.1172/JCI118402

Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies.

S C Liu 1, S J Yi 1, J R Mehta 1, P E Nichols 1, S K Ballas 1, P W Yacono 1, D E Golan 1, J Palek 1
PMCID: PMC507059  PMID: 8550846

Abstract

In red cells from patients with sickle cell anemia, hemoglobin S denatures and forms Heinz bodies. Binding of Heinz bodies to the inner surface of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3, outer surface-bound autologous IgG and, 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 fluorescence microscopy the incorporation and distribution of the fluorescent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phosphatidylserine and NBD-phosphatidylcholine in sickle red cells. Both phospholipid analogues are observed to accumulate prominently at sites of Heinz bodies. Accumulation at sites of Heinz bodies is also shown by 1,'1-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent lipid analogue that readily crosses membranes, but not by fluorescein-phosphatidylethanolamine, an analogue that is localized 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 lipids and proteins are sequestered.

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Selected References

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  1. Caulfield J. P., Chiang C. P., Yacono P. W., Smith L. A., Golan D. E. Low density lipoproteins bound to Schistosoma mansoni do not alter the rapid lateral diffusion or shedding of lipids in the outer surface membrane. J Cell Sci. 1991 May;99(Pt 1):167–173. doi: 10.1242/jcs.99.1.167. [DOI] [PubMed] [Google Scholar]
  2. Chiu D., Lubin B., Shohet S. B. Erythrocyte membrane lipid reorganization during the sickling process. Br J Haematol. 1979 Feb;41(2):223–234. doi: 10.1111/j.1365-2141.1979.tb05851.x. [DOI] [PubMed] [Google Scholar]
  3. Choe H. R., Schlegel R. A., Rubin E., Williamson P., Westerman M. P. Alteration of red cell membrane organization in sickle cell anaemia. Br J Haematol. 1986 Aug;63(4):761–773. doi: 10.1111/j.1365-2141.1986.tb07560.x. [DOI] [PubMed] [Google Scholar]
  4. Cobb C. E., Beth A. H. Identification of the eosinyl-5-maleimide reaction site on the human erythrocyte anion-exchange protein: overlap with the reaction sites of other chemical probes. Biochemistry. 1990 Sep 11;29(36):8283–8290. doi: 10.1021/bi00488a012. [DOI] [PubMed] [Google Scholar]
  5. Corbett J. D., Golan D. E. Band 3 and glycophorin are progressively aggregated in density-fractionated sickle and normal red blood cells. Evidence from rotational and lateral mobility studies. J Clin Invest. 1993 Jan;91(1):208–217. doi: 10.1172/JCI116172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Derick L. H., Liu S. C., Chishti A. H., Palek J. Protein immunolocalization in the spread erythrocyte membrane skeleton. Eur J Cell Biol. 1992 Apr;57(2):317–320. [PubMed] [Google Scholar]
  7. Dragsten P. R., Blumenthal R., Handler J. S. Membrane asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? Nature. 1981 Dec 24;294(5843):718–722. doi: 10.1038/294718a0. [DOI] [PubMed] [Google Scholar]
  8. Evans E., Mohandas N., Leung A. Static and dynamic rigidities of normal and sickle erythrocytes. Major influence of cell hemoglobin concentration. J Clin Invest. 1984 Feb;73(2):477–488. doi: 10.1172/JCI111234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Franck P. F., Bevers E. M., Lubin B. H., Comfurius P., Chiu D. T., Op den Kamp J. A., Zwaal R. F., van Deenen L. L., Roelofsen B. Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells. J Clin Invest. 1985 Jan;75(1):183–190. doi: 10.1172/JCI111672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Franck P. F., Chiu D. T., Op den Kamp J. A., Lubin B., van Deenen L. L., Roelofsen B. Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process. J Biol Chem. 1983 Jul 10;258(13):8436–8442. [PubMed] [Google Scholar]
  11. Golan D. E., Brown C. S., Cianci C. M., Furlong S. T., Caulfield J. P. Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components. J Cell Biol. 1986 Sep;103(3):819–828. doi: 10.1083/jcb.103.3.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hebbel R. P. Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood. 1991 Jan 15;77(2):214–237. [PubMed] [Google Scholar]
  13. Hebbel R. P. The sickle erythrocyte in double jeopardy: autoxidation and iron decompartmentalization. Semin Hematol. 1990 Jan;27(1):51–69. [PubMed] [Google Scholar]
  14. Kannan R., Labotka R., Low P. S. Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding. J Biol Chem. 1988 Sep 25;263(27):13766–13773. [PubMed] [Google Scholar]
  15. Kremer J. M., Esker M. W., Pathmamanoharan C., Wiersema P. H. Vesicles of variable diameter prepared by a modified injection method. Biochemistry. 1977 Aug 23;16(17):3932–3935. doi: 10.1021/bi00636a033. [DOI] [PubMed] [Google Scholar]
  16. Liu S. C., Derick L. H., Zhai S., Palek J. Uncoupling of the spectrin-based skeleton from the lipid bilayer in sickled red cells. Science. 1991 Apr 26;252(5005):574–576. doi: 10.1126/science.2020854. [DOI] [PubMed] [Google Scholar]
  17. Low P. S., Waugh S. M., Zinke K., Drenckhahn D. The role of hemoglobin denaturation and band 3 clustering in red blood cell aging. Science. 1985 Feb 1;227(4686):531–533. doi: 10.1126/science.2578228. [DOI] [PubMed] [Google Scholar]
  18. Lubin B., Chiu D., Bastacky J., Roelofsen B., Van Deenen L. L. Abnormalities in membrane phospholipid organization in sickled erythrocytes. J Clin Invest. 1981 Jun;67(6):1643–1649. doi: 10.1172/JCI110200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lutz H. U., Flepp R., Stringaro-Wipf G. Naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells. J Immunol. 1984 Nov;133(5):2610–2618. [PubMed] [Google Scholar]
  20. Lux S. E., John K. M., Karnovsky M. J. Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells. J Clin Invest. 1976 Oct;58(4):955–963. doi: 10.1172/JCI108549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Middelkoop E., Lubin B. H., Bevers E. M., Op den Kamp J. A., Comfurius P., Chiu D. T., Zwaal R. F., van Deenen L. L., Roelofsen B. Studies on sickled erythrocytes provide evidence that the asymmetric distribution of phosphatidylserine in the red cell membrane is maintained by both ATP-dependent translocation and interaction with membrane skeletal proteins. Biochim Biophys Acta. 1988 Jan 22;937(2):281–288. doi: 10.1016/0005-2736(88)90250-7. [DOI] [PubMed] [Google Scholar]
  22. Mohandas N., Rossi M., Bernstein S., Ballas S., Ravindranath Y., Wyatt J., Mentzer W. The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane. J Biol Chem. 1985 Nov 15;260(26):14264–14268. [PubMed] [Google Scholar]
  23. Nash G. B., Johnson C. S., Meiselman H. J. Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease. Blood. 1984 Jan;63(1):73–82. [PubMed] [Google Scholar]
  24. Nash G. B., Wyard S. J. Changes in surface area and volume measured by micropipette aspiration for erythrocytes ageing in vivo. Biorheology. 1980;17(5-6):479–484. [PubMed] [Google Scholar]
  25. Platt O. S., Falcone J. F., Lux S. E. Molecular defect in the sickle erythrocyte skeleton. Abnormal spectrin binding to sickle inside-our vesicles. J Clin Invest. 1985 Jan;75(1):266–271. doi: 10.1172/JCI111684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rifkind R. A. Heinz body anemia: an ultrastructural study. II. Red cell sequestration and destruction. Blood. 1965 Oct;26(4):433–448. [PubMed] [Google Scholar]
  27. Schlegel R. A., Phelps B. M., Waggoner A., Terada L., Williamson P. Binding of merocyanine 540 to normal and leukemic erythroid cells. Cell. 1980 Jun;20(2):321–328. doi: 10.1016/0092-8674(80)90618-2. [DOI] [PubMed] [Google Scholar]
  28. Schlüter K., Drenckhahn D. Co-clustering of denatured hemoglobin with band 3: its role in binding of autoantibodies against band 3 to abnormal and aged erythrocytes. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6137–6141. doi: 10.1073/pnas.83.16.6137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sims P. J., Waggoner A. S., Wang C. H., Hoffman J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. doi: 10.1021/bi00713a022. [DOI] [PubMed] [Google Scholar]
  30. Stillwell W., Wassall S. R., Dumaual A. C., Ehringer W. D., Browning C. W., Jenski L. J. Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells. Biochim Biophys Acta. 1993 Feb 23;1146(1):136–144. doi: 10.1016/0005-2736(93)90348-4. [DOI] [PubMed] [Google Scholar]
  31. Struck D. K., Pagano R. E. Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J Biol Chem. 1980 Jun 10;255(11):5404–5410. [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Turrini F., Arese P., Yuan J., Low P. S. Clustering of integral membrane proteins of the human erythrocyte membrane stimulates autologous IgG binding, complement deposition, and phagocytosis. J Biol Chem. 1991 Dec 15;266(35):23611–23617. [PubMed] [Google Scholar]
  34. Turrini F., Mannu F., Arese P., Yuan J., Low P. S. Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins. Blood. 1993 Jun 1;81(11):3146–3152. [PubMed] [Google Scholar]
  35. Waugh S. M., Low P. S. Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane. Biochemistry. 1985 Jan 1;24(1):34–39. doi: 10.1021/bi00322a006. [DOI] [PubMed] [Google Scholar]
  36. Waugh S. M., Willardson B. M., Kannan R., Labotka R. J., Low P. S. Heinz bodies induce clustering of band 3, glycophorin, and ankyrin in sickle cell erythrocytes. J Clin Invest. 1986 Nov;78(5):1155–1160. doi: 10.1172/JCI112696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Williamson P. L., Massey W. A., Phelps B. M., Schlegel R. A. Membrane phase state and the rearrangement of hematopoietic cell surface receptors. Mol Cell Biol. 1981 Feb;1(2):128–135. doi: 10.1128/mcb.1.2.128. [DOI] [PMC free article] [PubMed] [Google Scholar]

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