The Journal of Membrane Biology

, Volume 246, Issue 8, pp 591–607 | Cite as

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

  • Savita Sharma
  • Surekha M. Zingde
  • Sadashiv M. Gokhale
Article
First Online:
Received:
Accepted:

Abstract

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.

Keywords

Erythrocyte Membrane skeleton Heat stress Electrophoresis Mass spectrometry 
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Notes

Acknowledgments

We thank Mr. S. Dolas, from the mass spectroscopy facility, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai, for his help and suggestions. We also thank all those who donated blood for this study. This work was supported by a grant to S. M. G. from the University Grants Commission, New Delhi, India [Major Research Project F-37-138/2009 (SR)].

References

  1. Amer J, Zelig O, Fibach E (2008) Oxidative status of red blood cells, neutrophils, and platelets in paroxysmal nocturnal hemoglobinuria. Exp Hematol 36:369–377PubMedCrossRefGoogle Scholar
  2. Becker PS, Cohen CM, Lux SE (1986) The effect of mild diamide oxidation on the structure and function of human erythrocyte spectrin. J Biol Chem 261:4620–4628PubMedGoogle Scholar
  3. Bennett V (1990) Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev 70:1029–1065PubMedGoogle Scholar
  4. Biondani A, Franco T, Franco C, Alessandro M, Alida F, Angela S, Yves B, Lucia De F (2008) Heat-shock protein-27, -70 and peroxiredoxin-II show molecular chaperone function in sickle red cells: evidence from transgenic sickle cell mouse model. Proteomics Clin Appl 2:706–719PubMedCrossRefGoogle Scholar
  5. Brandts JF, Erickson L, Lysko K, Schwartz AT, Taverna RD (1977) Calorimetric studies of the structural transitions of the human erythrocyte membrane. The involvement of spectrin in the A transition. Biochemistry 16:3450–3454PubMedCrossRefGoogle Scholar
  6. Coakley WT, Bater AJ, Crum LA, Deeley JO (1979) Morphological changes, haemolysis and microvesicularization of heated human erythrocytes. J Therm Biol 4:85–93CrossRefGoogle Scholar
  7. D’Amici GM, Rinalducci S, Zolla L (2007) Proteomic analysis of RBC membrane protein degradation during blood storage. J Proteome Res 6:3242–3255PubMedCrossRefGoogle Scholar
  8. Devasena T, Lalitha S, Padma K (2001) Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis. Clin Chim Acta 308:155–161PubMedCrossRefGoogle Scholar
  9. Eskelinen S, Coakley WT, Tilley D (1985) Thermal denaturation of the erythrocyte cytoskeleton alters the morphological changes associated with osmotic swelling. J Therm Biol 10:187–190CrossRefGoogle Scholar
  10. Goodman SR, Kurdia A, Ammann L, Kakhniashvili D, Daescu O (2007) The human red blood cell proteome and interactome. Exp Biol Med 232:1391–1408CrossRefGoogle Scholar
  11. Gudi T, Gupta CM (1993) Hsp 70-like protein in rhesus erythrocyte cytosol and its interactions with membrane skeleton under heat and pathologic stress. J Biol Chem 268:21344–21350PubMedGoogle Scholar
  12. Gudi SRP, Kumar A, Bhakuni V, Gokhale SM, Gupta CM (1990) Membrane skeleton–bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes. Biochim Biophys Acta 1023:63–72PubMedCrossRefGoogle Scholar
  13. Halliwell B, Gutteridge JMC (1986) Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 246:501–514PubMedCrossRefGoogle Scholar
  14. Hanahan DJ, Ekholm JE (1974) The preparation of red cell ghosts (membranes). Methods Enzymol 31:168–172PubMedCrossRefGoogle Scholar
  15. Ho KC, Lin PS (1991) Response of erythrocytes to heat in the presence of D2O, glycerol, and anisotonic saline. Radiat Res 125:20–27PubMedCrossRefGoogle Scholar
  16. Houston H, Mearow KM (1979) Temperature-related changes in the erythrocytic carbonic anhydrase (actazolamide-sensitive estrase) activity of goldfish, Carassius auratus. J Exp Biol 78:255–264PubMedGoogle Scholar
  17. Jaja SI, Ikotum AR, Gbenebitse S, Temiye EO (2002) Blood pressure, hematologic and erythrocyte fragility changes in children suffering from sickle cell anemia following ascorbic acid supplementation. J Trop Pediatr 48:366–370PubMedCrossRefGoogle Scholar
  18. Jozwiak Z, Palecz D, Leyko W (1991) The response of pig erythrocytes to thermal stress. Int J Radiat Biol 59:479–487PubMedCrossRefGoogle Scholar
  19. Kakhniashvili DG, Bulla LA Jr, Goodman SR (2004) The human erythrocyte proteome: analysis by ion trap mass spectrometry. Mol Cell Proteomics 3:501–509PubMedCrossRefGoogle Scholar
  20. Knowles WJ, Morrow JS, Speicher DW, Zarkowsky HS, Mohandas N, Mentzer WC, Shohet SB, Marchesi VT (1983) Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis. J Clin Invest 71:1867–1877PubMedCrossRefGoogle Scholar
  21. Kriebardis AG, Antonelou MH, Stamoulis KS, Economou-Petersen E, Margaritis LH, Papassideri IS (2007) Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells. J Cell Mol Med 11:148–155PubMedCrossRefGoogle Scholar
  22. Kumar A, Gudi SR, Gokhale SM, Bhakuni V, Gupta CM (1990) Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions. Biochim Biophys Acta 1030:269–278PubMedCrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  24. Lepock JR, Frey HE, Bayne H, Markus J (1989) Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins. Biochim Biophys Acta 14:191–201Google Scholar
  25. Lii CK, Hung CN (1997) Protein thiol modifications of human red blood cells treated with t-butyl hydroperoxide. Biochim Biophys Acta 1336:147–156PubMedCrossRefGoogle Scholar
  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  27. Lysko KA, Carlson R, Taverna R, Snow J, Brandts JF (1981) Protein involvement in structural transitions of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation. Biochemistry 20:5570–5576PubMedCrossRefGoogle Scholar
  28. Minetti M, Ceccarini M, Distasi AMM, Petrucci TC, Marchesi VT (1986) Spectrin involvement in a 40°C structural transition of the red blood cell membrane. J Cell Biochem 30:361–370PubMedCrossRefGoogle Scholar
  29. Mohandas N, Gallagher PG (2008) Red cell membrane: past, present, and future. Blood 112:3939–3948PubMedCrossRefGoogle Scholar
  30. Ozturk O, Gumuslu S (2004) Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress. Life Sci 75:1551–1565PubMedCrossRefGoogle Scholar
  31. Pasini EM, Kirkegaard M, Mortensen P, Lutz HU (2006) In-depth analysis of the membrane and cytosolic proteome of red blood cells. Blood 108:791–801PubMedCrossRefGoogle Scholar
  32. Rocha S, Costa E, Coimbra S, Nascimento H, Catarino C, Rocha-Pereira P, Quintanilha A, Belo L, Santos-Silva A (2009) Linkage of cytosolic peroxiredoxin 2 to erythrocyte membrane imposed by hydrogen peroxide–induced oxidative stress. Blood Cells Mol Dis 43:68–73PubMedCrossRefGoogle Scholar
  33. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858PubMedCrossRefGoogle Scholar
  34. Smith DK, Palek J (1983) Sulfhydryl reagents induce altered spectrin self-association, skeletal instability and increased thermal sensitivity of red cells. Blood 62:1190–1196PubMedGoogle Scholar
  35. Streichman S, Hertz E, Tatarsky I (1988) Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association. Biochim Biophys Acta 942:333–340PubMedCrossRefGoogle Scholar
  36. Utoh J, Zajkowski B, Joy E, Harasaki H (1992) Effects of heat on fragility and morphology of human and calf erythrocytes. J Invest Surg 5:305–313PubMedCrossRefGoogle Scholar
  37. Wagner CT, Lu IY, Hoffman MH, Sun WQ, Trent JD, Connor J (2004) T-complex polypeptide-1 interacts with the erythrocyte cytoskeleton in response to elevated temperatures. J Biol Chem 279:16223–16228PubMedCrossRefGoogle Scholar
  38. Yoshino H, Marchesi VT (1984) Isolation of spectrin subunits and reassociation in vitro. Analysis by fluorescence polarization. J Biol Chem 259:4496–4500PubMedGoogle Scholar
  39. Yoshino H, Minari O (1987) Heat induced dissociation of human erythrocyte spectrin dimer into monomers. Biochim Biophys Acta 905:100–108PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Savita Sharma
    • 1
  • Surekha M. Zingde
    • 2
  • Sadashiv M. Gokhale
    • 1
  1. 1.School of Biochemistry, Devi Ahilya UniversityIndoreIndia
  2. 2.Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial CentreKhargharIndia

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