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Logo of jcinvestThe Journal of Clinical Investigation
J Clin Invest. 1988 March; 81(3): 893–901.
PMCID: PMC442542

Deficiency of protein 4.2 in erythrocytes from a patient with a Coombs negative hemolytic anemia. Evidence for a role of protein 4.2 in stabilizing ankyrin on the membrane.


A patient with a mild hemolytic anemia and osmotically fragile, spherocytic erythrocytes was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of protein 4.2 (less than 0.10% of normal). The protein 4.2-deficient erythrocytes contained normal amounts of all other membrane proteins, although the amount of band 3 was slightly reduced and the amount of band 6 (G3PD) was slightly elevated. The spectrin content of these cells was normal, as measured by both SDS-PAGE and radioimmunoassay. Erythrocytes from the patient's biologic parents were hematologically normal and contained normal amounts of protein 4.2. Immunological analysis using affinity purified antibodies revealed that the patient's protein 4.2 was composed of equal amounts of a 74-kD and 72-kD protein doublet, whereas the normal protein was composed primarily of a 72-kD monomer. Proteolytic digestion studies using trypsin, alpha-chymotrypsin and papain demonstrated that the patient's protein 4.2 was similar but not identical to the normal protein. Binding studies showed that the protein 4.2-deficient membranes bound purified protein 4.2 to the same extent as normal membranes, suggesting that the membrane binding site(s) for the protein were normal. Depleting the protein 4.2-deficient membranes of spectrin and actin resulted in a loss of nearly two-thirds of the membrane ankyrin, whereas similar depletion of normal membranes resulted in no loss of ankyrin. Repletion of the protein 4.2-deficient membranes with purified protein 4.2 before spectrin-actin extraction partially prevented the loss of ankyrin. These results suggest that protein 4.2 may function to stabilize ankyrin on the erythrocyte membrane.

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Selected References

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  • Goodman SR, Shiffer K. The spectrin membrane skeleton of normal and abnormal human erythrocytes: a review. Am J Physiol. 1983 Mar;244(3):C121–C141. [PubMed]
  • Agre P, Casella JF, Zinkham WH, McMillan C, Bennett V. Partial deficiency of erythrocyte spectrin in hereditary spherocytosis. Nature. 314(6009):380–383. [PubMed]
  • Wolfe LC, John KM, Falcone JC, Byrne AM, Lux SE. A genetic defect in the binding of protein 4.1 to spectrin in a kindred with hereditary spherocytosis. N Engl J Med. 1982 Nov 25;307(22):1367–1374. [PubMed]
  • Tchernia G, Mohandas N, Shohet SB. Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability. J Clin Invest. 1981 Aug;68(2):454–460. [PMC free article] [PubMed]
  • Liu SC, Palek J, Prchal JT. Defective spectrin dimer-dimer association with hereditary elliptocytosis. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2072–2076. [PubMed]
  • Steck TL. Cross-linking the major proteins of the isolated erythrocyte membrane. J Mol Biol. 1972 May 14;66(2):295–305. [PubMed]
  • Bennett V, Stenbuck PJ. The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes. Nature. 1979 Aug 9;280(5722):468–473. [PubMed]
  • Mohandas N, Clark MR, Jacobs MS, Shohet SB. Analysis of factors regulating erythrocyte deformability. J Clin Invest. 1980 Sep;66(3):563–573. [PMC free article] [PubMed]
  • ZLATKIS A, ZAK B, BOYLE AJ. A new method for the direct determination of serum cholesterol. J Lab Clin Med. 1953 Mar;41(3):486–492. [PubMed]
  • BARTLETT GR. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed]
  • DODGE JT, MITCHELL C, HANAHAN DJ. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. [PubMed]
  • Bennett V, Branton D. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin. J Biol Chem. 1977 Apr 25;252(8):2753–2763. [PubMed]
  • Hargreaves WR, Giedd KN, Verkleij A, Branton D. Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles. J Biol Chem. 1980 Dec 25;255(24):11965–11972. [PubMed]
  • Tyler JM, Reinhardt BN, Branton D. Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin. J Biol Chem. 1980 Jul 25;255(14):7034–7039. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Fairbanks G, Steck TL, Wallach DF. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. [PubMed]
  • 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. [PubMed]
  • Korsgren C, Cohen CM. Purification and properties of human erythrocyte band 4.2. Association with the cytoplasmic domain of band 3. J Biol Chem. 1986 Apr 25;261(12):5536–5543. [PubMed]
  • Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. [PubMed]
  • Steck TL. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. [PMC free article] [PubMed]
  • Palek J, Liu SC, Liu PY, Prchal J, Castleberry RP. Altered assembly of spectrin in red cell membranes in hereditary pyropoikilocytosis. Blood. 1981 Jan;57(1):130–139. [PubMed]
  • Hayashi S, Koomoto R, Yano A, Ishigami S, Tsujino G. Abnormality in a specific protein of the erythrocyte membrane in hereditary spherocytosis. Biochem Biophys Res Commun. 1974 Apr 23;57(4):1038–1044. [PubMed]
  • Nozawa Y, Noguchi T, Iida H, Fukushima H, Sekiya T. Erythrocyte membrane of hereditary spherocytosis: alteration in surface ultrastructure and membrane proteins, as inferred by scanning electron microscopy and SDS-disc gel electrophoresis. Clin Chim Acta. 1974 Aug 30;55(1):81–85. [PubMed]
  • Iida H, Hasegawa I, Nozawa Y. Biochemical studies on abnormal erythrocyte membranes. Protein abnormality of erythrocyte membrane in biliary obstruction. Biochim Biophys Acta. 1976 Sep 7;443(3):394–401. [PubMed]
  • Bennett V, Stenbuck PJ. Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane. J Biol Chem. 1980 Jul 10;255(13):6424–6432. [PubMed]

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