Skip to main content

Advertisement

SpringerLink
Log in

Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-β, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Morton NE, MacKinney AA, Kosower N (1962) Genetics of spherocytosis. Am J Hum Genet 14:170–178

    PubMed  CAS  Google Scholar 

  2. Godal HC, Heisto H (1981) High prevalence of increased osmotic fragility of red blood cells among Norwegian blood donors. Scand J Haematol 27:30–34

    Article  PubMed  CAS  Google Scholar 

  3. Tse WT, Lux SE (1999) Red blood cell membrane disorders. Br J Haematol 104:2–13

    Article  PubMed  CAS  Google Scholar 

  4. Eber SW, Pekrun A, Neufeldt A, Schroter W (1992) Prevalence of increased osmotic fragility of erythrocytes in German blood donors: screening using a modified glycerol lysis test. Ann Hematol 64:88–92

    Article  PubMed  CAS  Google Scholar 

  5. Delaunay J (2007) The molecular basis of hereditary red cell membrane disorders. Blood Rev 21:1–20

    Article  PubMed  CAS  Google Scholar 

  6. Eber SW, Armbrust R, Schofer W (1990) Variable clinical severity of hereditary spherocytosis: relation to erythrocyte spectrin concentration, osmotic fragility, and autohemolysis. J Pediatr 117:409–416

    Article  PubMed  CAS  Google Scholar 

  7. Del Giudice EM, Perrotta S, Nobili B, Pinto L, Cutillo L, Iolascon A (1993) Coexistence of hereditary spherocytosis (HS) due to band 3 deficiency and β thalassemia trait; partial correction HS of phenotype. Br J Haematol 85:553–557

    Article  Google Scholar 

  8. Del Giudice EM, Iolascon A, Pinto L, Nobili B, Perrotta S (1994) Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis. Br J Haematol 88:52–55

    Article  Google Scholar 

  9. Bolton-Maggs PH, Stevens RF, Dodd NJ, Lamont G, Tittensor P, King MJ (2004) Guidelines for the diagnosis and management of hereditary spherocytosis. Br J Haematol 126:455–474

    Article  PubMed  CAS  Google Scholar 

  10. Perrotta S, Gallagher PG, Mohandas N (2008) Hereditary spherocytosis. Lancet 372:1411–1426

    Article  PubMed  CAS  Google Scholar 

  11. Cooper RA, Jandl JH (1969) The role of membrane lipids in the survival of red cells in hereditary spherocytosis. J Clin Investig 48:736–744

    Article  PubMed  CAS  Google Scholar 

  12. Da Costa L, Mohandas N, Sorette M, Grange MJ, Tchernia G, Cynober T (2001) Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia. Blood 98:2894–2899

    Article  PubMed  CAS  Google Scholar 

  13. Segel GB (2004) Hereditary spherocytosis. In: Behrman RE, Kliegman RM, Jenson HB (eds) Nelson’s textbook of pediatrics, 17th edn. Saunders, Philadelphia, pp 1620–1621

    Google Scholar 

  14. Jarolim P, Murray JL, Rubin HL et al (1996) Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency. Blood 88:4366–4374

    PubMed  CAS  Google Scholar 

  15. Agre P, Asimos A, Casella JF, McMillan C (1986) Inheritance pattern and clinical response to splenectomy as a reflection of erythrocyte spectrin deficiency in hereditary spherocytosis. N Engl J Med 315:1579–1583

    Article  PubMed  CAS  Google Scholar 

  16. Nakanishi H, Kanzaki A, Yawata A, Yamada O, Yawata Y (2001) Ankyrin gene mutations in Japanese patients with hereditary spherocytosis. Int J Hematol 73:54–63

    Article  PubMed  CAS  Google Scholar 

  17. Weidekamm E, Brdiczka B, Wildermuth M (1978) Phospholipid composition of human erythrocyte spectrin. Mol Biol Rep 4:25–28

    Article  PubMed  CAS  Google Scholar 

  18. Inoue T, Kanzaki A, Yawata A et al (1994) Uniquely higher incidence of isolated or combined deficiency of band 3 and/or band 4.2 as the pathogenesis of autosomal dominantly inherited hereditary spherocytosis in the Japanese population. Int J Hematol 60:227–238

    PubMed  CAS  Google Scholar 

  19. Mariani M, Barcellini W, Vercellati C et al (2008) Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect. Haematologica 93:1310–1317

    Article  PubMed  CAS  Google Scholar 

  20. Hanspal M, Yoon SH, Yu H, Hanspal JS, Lambert S, Palek J, Prchal JT (1991) Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin. Blood 77:165–173

    PubMed  CAS  Google Scholar 

  21. Lanciotti M, Perutelli P, Valetto A, Di Martino D, Mori PG (1997) Ankyrin deficiency is the most common defect in dominant and nondominant hereditary spherocytosis. Haematologica 82:460–462

    PubMed  CAS  Google Scholar 

  22. Miraglia del Giudice E, Francese M, Polito R, Nobili B, Iolascon A, Perrotta S (1997) Apparently normal ankyrin content in unsplenectomized hereditary spherocytosis patients with the inactivation of one ankyrin (ANK1) allele. Haematologica 82:332–333

    PubMed  CAS  Google Scholar 

  23. An X, Mohandas N (2008) Disorders of red cell membrane. Br J Haematol 141:367–375

    PubMed  CAS  Google Scholar 

  24. Cynober T, Mohandas N, Tchernia G (1996) Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. J Lab Clin Med 128:259–269

    Article  PubMed  CAS  Google Scholar 

  25. Ricard MP, Gilsanz F, Millan I (2000) Erythroid membrane protein defects in hereditary spherocytosis. A study of 62 Spanish cases. Haematologica 85:994–995

    PubMed  CAS  Google Scholar 

  26. Lee YK, Cho HI, Park SS, Lee YJ, Ra E, Chang YH et al (2000) Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis. J Korean Med Sci 15:284–288

    PubMed  CAS  Google Scholar 

  27. Iolascon A, Perrotta S, Stewart GW (2003) Red blood cell membrane defects. Rev Clin Exp Hematol 7:22–56

    PubMed  CAS  Google Scholar 

  28. Low TY, Seow TK, Chung MC (2002) Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2:1229–1239

    Article  PubMed  CAS  Google Scholar 

  29. Nowacka M, Jackowiak P, Rybarczyk A, Magacz T, Strozycki PM, Barciszewski J, Figlerowicz M (2011) 2D-PAGE as an effective method of RNA degradome analysis. Mol Biol Rep. doi:10.1007/s11033-011-0718-1

  30. Liu W, Ma Y, Huang L, Peng J, Zhang P, Zhang H, Chen J, Qin H (2010) Identification of HSP27 as a potential tumor marker for colorectal cancer by the two-dimensional polyacrylamide gel electrophoresis. Mol Biol Rep 37:3207–3216

    Article  PubMed  CAS  Google Scholar 

  31. Li C, Tan YX, Zhou H, Ding SJ, Li SJ, Ma DJ, Man XB et al (2005) Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma: identification of potential tumor markers. Proteomics 5:1125–1139

    Article  PubMed  Google Scholar 

  32. Petricoin EF, Zoon KC, Kohn EC, Barrett JC, Liotta LA (2002) Clinical proteomics: translating benchside promise into bedside reality. Nat Rev Drug Discov 1:683–695

    Article  PubMed  CAS  Google Scholar 

  33. Li Z, Zhao X, Bai S, Wang Z, Chen L, Wei Y, Huang C (2008) Proteomics identification of cyclophilin A as a potential prognostic factor and therapeutic target in endometrial carcinoma. Mol Cell Proteomics 7:1810–1823

    Article  PubMed  CAS  Google Scholar 

  34. Herrmann PC, Liotta LA, Petricoin EF III (2001) Cancer proteomics: the state of the art. Dis Markers 17:49–57

    PubMed  CAS  Google Scholar 

  35. Bruschi M, Seppi C, Arena S et al (2005) Proteomic analysis of erythrocyte membranes by soft immobiline gels combined with differential protein extraction. J Proteome Res 4:1304–1309

    Article  PubMed  CAS  Google Scholar 

  36. Rabilloud T (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2:3–10

    Article  PubMed  CAS  Google Scholar 

  37. Friedman DB, Hill S, Keller JW, Merchant NB, Levy SE, Coffey RJ, Caprioli RM (2004) Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 4:793–811

    Article  PubMed  CAS  Google Scholar 

  38. Dodge JT, Mitchell C, Hanahan DJ (1963) The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119–130

    Article  PubMed  CAS  Google Scholar 

  39. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  40. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  PubMed  CAS  Google Scholar 

  41. Bhakdi S, Knufermann H, Wallach DF (1975) Two-dimensional separation of erythrocyte membrane proteins. Biochim Biophys Acta 394:550–557

    Article  PubMed  CAS  Google Scholar 

  42. Anselstetter V, Horstmann HJ (1975) Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane. Eur J Biochem 56:259–269

    Article  PubMed  CAS  Google Scholar 

  43. Rubin RW, Milikowski C Over two hundred polypeptides resolved from the human erythrocyte membrane. Biochim Biophys Acta 509:100–110

  44. Harell D, Morrison M (1979) Two-dimensional separation of erythrocyte membrane proteins. Arch Biochem Biophys 193:158–168

    Article  PubMed  CAS  Google Scholar 

  45. Copeland BR, Todd SA, Furlong CE (1982) High resolution two dimensional gel electrophoresis of human erythrocyte membrane proteins. Am J Hum Genet 34:15–31

    PubMed  CAS  Google Scholar 

  46. Heegaard NHH, Poglod R (1991) Separation of normal human erythrocyte membrane proteins by high resolution two-dimensional gel electrophoresis. Appl Theor Electrophor 2:109–127

    PubMed  CAS  Google Scholar 

  47. Rabilloud T, Blisnick T, Heller M, Luche S et al (1999) Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum—infected erythrocyte ghosts. Electrophoresis 20:3603–3610

    Article  PubMed  CAS  Google Scholar 

  48. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021

    PubMed  Google Scholar 

  49. O’Farrell PZ, Goodman HM, O’Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1142

    Article  PubMed  Google Scholar 

  50. Rubin RW, Milikowski C (1978) Over two hundred polypeptides resolved from the human erythrocyte membrane. Biochim Biophys Acta 509:100–110

    Article  PubMed  CAS  Google Scholar 

  51. Rosenblum BB, Furlong CE, Stamatoyannopoulos G (1979) Two dimensional electrophoresis of red cell membrane proteins. Am J Hum Genet 31:60A

    Google Scholar 

  52. Anderson NG, Anderson NL (1978) Analytical techniques for cell fractions. XXI. Two dimensional analysis of serum and tissue proteins. Multiple isoelectric focusing. Anal Biochem 85:331–340

    Article  PubMed  CAS  Google Scholar 

  53. Fairbanks G, Steck TL, Wallach DFH (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617

    Article  PubMed  CAS  Google Scholar 

  54. Switzer RC III, Merril CR, Shifrin S (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237

    Article  PubMed  CAS  Google Scholar 

  55. Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363

    Article  PubMed  CAS  Google Scholar 

  56. Steck TL, Kant JA (1974) Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol 31A:172–180

    Article  Google Scholar 

  57. Ames GF-L, Nikaido K (1976) Two-dimensional gel electrophoresis of membrane proteins. Biochemistry 15:616–623

    Article  PubMed  CAS  Google Scholar 

  58. Caterino M, Ruoppolo M, Orrù S, Savoia M, Perrotta S, Del Vecchio L, Salvatore F, Stewart GW, Iolascon A (2006) Characterization of red cell membrane proteins as a function of red cell density: annexin VII in different forms of hereditary spherocytosis. FEBS Lett 580:6527–6532

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Ankara University Biotechnology Institute, Ankara, Turkey

    Selen Peker

  2. Pediatric Molecular Genetics Department, Medical School, Ankara University, Ankara, Turkey

    Nejat Akar

  3. Ankara University Biotechnology Institute, Central Laboratory, Proteomics Department, 35110, Tandogan-Ankara, Turkey

    Duygu Ozel Demiralp

Authors
  1. Selen Peker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nejat Akar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Duygu Ozel Demiralp
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Duygu Ozel Demiralp.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 39 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Peker, S., Akar, N. & Demiralp, D.O. Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis. Mol Biol Rep 39, 3161–3167 (2012). https://doi.org/10.1007/s11033-011-1082-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-1082-x

Keywords

Search

Navigation