Cell Stress and Chaperones

, Volume 17, Issue 2, pp 215–227 | Cite as

Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects, patients with coronary heart disease and inflammatory bowel disease

  • George Füst
  • Katalin Uray
  • László Bene
  • Ferenc Hudecz
  • István Karádi
  • Zoltán Prohászka
Original Paper


Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.


Coronary heart disease Inflammatory bowel disease Human HSP60 Mycobacterial HSP65 Epitope analysis 

Supplementary material

12192_2011_301_MOESM1_ESM.doc (303 kb)
Online Table 1(DOC 303 kb)
12192_2011_301_MOESM2_ESM.ppt (132 kb)
Online Fig. 1Three-dimensional model of human Hsp60 monomer protein based on the tertiary structure of E. coli Hsp60. a The sequences marked by the ribbon structure represent a sequences recognized by IBD patients’ sera on the HSP60 set of pin-bound peptides, numbered according to human HSP60, b sequences recognized by IBD patients’ sera on the HSP65 set of pin-bound peptides, numbered according to M. bovis HSP65, and c sequences recognized on the HSP65, but not on the HSP60 set of pin-bound peptides, numbered according to M. bovis HSP65. The insert shows the localization of a GroEL monomer in the oligomeric protein structure of GroEL–GroES machinery consisting of 14 GroEL monomers and an additional ring of seven GroES monomers (cap). The colors of the ribbon structure represent the localization of the peptide within the linear sequence of the protein, from the N-terminal (dark blue) till the C-terminal (red). (PPT 132 kb)
12192_2011_301_MOESM3_ESM.ppt (140 kb)
Online Fig. 2HSP60- and HSP65-dominant regions with HP, IBD, and CHD sera: the colors of the ribbon structure indicate the accessibility of the residues according to the Swiss-PdbViewer (GlaxoSmithKline), dark blue buried, red exposed. Differential recognition of HSP65 peptides by HP and IBD sera is marked with asterisks (PPT 139 kb)


  1. Ayada K, Yokota K, Kobayashi K, Shoenfeld Y, Matsuura E, Oguma K (2009) Chronic infections and atherosclerosis. Clin Rev Allergy Immunol 37:44–48PubMedCrossRefGoogle Scholar
  2. Bene L, Fust G, Huszti Z, Hernadi Z, Fekete B, Meszaros M, Veres A, Kovacs A, Miklos K, Singh M, Romics L, Prohaszka Z (2002) Impaired humoral immune response against mycobacterial 65-kDa heat shock protein (HSP65) in patients with inflammatory bowel disease. Dig Dis Sci 47:1432–1437PubMedCrossRefGoogle Scholar
  3. Birnie DH, Holme ER, McKay IC, Hood S, McColl KE, Hillis WS (1998) Association between antibodies to heat shock protein 65 and coronary atherosclerosis. Possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk. Eur Heart J 19:387–394PubMedCrossRefGoogle Scholar
  4. Birnie DH, Vickers LE, Hillis WS, Norrie J, Cobbe SM (2005) Increased titres of anti-human heat shock protein 60 predict an adverse one year prognosis in patients with acute cardiac chest pain. Heart 91:1148–1153PubMedCrossRefGoogle Scholar
  5. Burian K, Kis Z, Virok D, Endresz V, Prohaszka Z, Duba J, Berencsi K, Boda K, Horvath L, Romics L, Fust G, Gonczol E (2001) Independent and joint effects of antibodies to human heat-shock protein 60 and Chlamydia pneumoniae infection in the development of coronary atherosclerosis. Circulation 103:1503–1508PubMedGoogle Scholar
  6. Campanella C, Marino Gammazza A, Mularoni L, Cappello F, Zummo G, Di Felice V (2009) A comparative analysis of the products of GROEL-1 gene from Chlamydia trachomatis serovar D and the HSP60 var1 transcript from Homo sapiens suggests a possible autoimmune response. Int J Immunogenet 36:73–78PubMedCrossRefGoogle Scholar
  7. Cohen IR, Young DB (1991) Autoimmunity, microbial immunity and the immunological homunculus. Immunol Today 12:105–110PubMedCrossRefGoogle Scholar
  8. Foteinos G, Afzal AR, Mandal K, Jahangiri M, Xu Q (2005) Anti-heat shock protein 60 autoantibodies induce atherosclerosis in apolipoprotein E-deficient mice via endothelial damage. Circulation 112:1206–1213PubMedCrossRefGoogle Scholar
  9. George J, Afek A, Gilburd B, Shoenfeld Y, Harats D (2001) Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice. J Am Coll Cardiol 38:900–905PubMedCrossRefGoogle Scholar
  10. Geysen HM, Meloen RH, Barteling SJ (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci U S A 81:3998–4002PubMedCrossRefGoogle Scholar
  11. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723PubMedCrossRefGoogle Scholar
  12. Guex N, Diemand A, Peitsch MC (1999) Protein modelling for all. Trends Biochem Sci 24:364–367PubMedCrossRefGoogle Scholar
  13. Harats D, Yacov N, Gilburd B, Shoenfeld Y, George J (2002) Oral tolerance with heat shock protein 65 attenuates Mycobacterium tuberculosis-induced and high-fat-diet-driven atherosclerotic lesions. J Am Coll Cardiol 40:1333–1338PubMedCrossRefGoogle Scholar
  14. Harkonen T, Puolakkainen M, Sarvas M, Airaksinen U, Hovi T, Roivainen M (2000) Picornavirus proteins share antigenic determinants with heat shock proteins 60/65. J Med Virol 62:383–391PubMedCrossRefGoogle Scholar
  15. Hoppichler F, Lechleitner M, Traweger C, Schett G, Dzien A, Sturm W, Xu Q (1996) Changes of serum antibodies to heat-shock protein 65 in coronary heart disease and acute myocardial infarction. Atherosclerosis 126:333–338PubMedCrossRefGoogle Scholar
  16. Horvath L, Cervenak L, Oroszlan M, Prohaszka Z, Uray K, Hudecz F, Baranyi E, Madacsy L, Singh M, Romics L, Fust G, Panczel P (2002) Antibodies against different epitopes of heat-shock protein 60 in children with type 1 diabetes mellitus. Immunol Lett 80:155–162PubMedCrossRefGoogle Scholar
  17. Huszti Z, Bene L, Kovacs A, Fekete B, Fust G, Romics L, Singh M, Prohaszka Z (2004) Low levels of antibodies against E. coli and mycobacterial 65 kDa heat shock proteins in patients with inflammatory bowel disease. Inflamm Res 53:551–555PubMedCrossRefGoogle Scholar
  18. Kocsis J, Prohaszka Z, Biro A, Fust G, Banhegyi D (2003) Elevated levels of antibodies against 70 kDa heat shock proteins in the sera of patients with HIV infection. J Med Virol 71:480–482PubMedCrossRefGoogle Scholar
  19. Krchnak V, Vagner J, Lebl M (1988) Noninvasive continuous monitoring of solid-phase peptide synthesis by acid–base indicator. Int J Pept Protein Res 32:415–416PubMedCrossRefGoogle Scholar
  20. Lachumanan R, Devi S, Cheong YM, Rodda SJ, Pang T (1993) Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi. Infect Immun 61:4527–4531PubMedGoogle Scholar
  21. Lindquist S, Craig EA (1988) The heat-shock proteins. Annu Rev Genet 22:631–677PubMedCrossRefGoogle Scholar
  22. Lu X, Chen D, Endresz V, Xia M, Faludi I, Burian K, Szabo A, Csanadi A, Miczak A, Gonczol E, Kakkar V (2010) Immunization with a combination of ApoB and HSP60 epitopes significantly reduces early atherosclerotic lesion in Apobtm2SgyLdlrtm1Her/J mice. Atherosclerosis 212:472–480PubMedCrossRefGoogle Scholar
  23. Maeda H, Miyamoto M, Kokeguchi S, Kono T, Nishimura F, Takashiba S, Murayama Y (2000) Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis. FEMS Immunol Med Microbiol 28:219–224PubMedCrossRefGoogle Scholar
  24. Maron R, Sukhova G, Faria AM, Hoffmann E, Mach F, Libby P, Weiner HL (2002) Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice. Circulation 106:1708–1715PubMedCrossRefGoogle Scholar
  25. Metzler B, Schett G, Kleindienst R, van der Zee R, Ottenhoff T, Hajeer A, Bernstein R, Xu Q, Wick G (1997) Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis. Arterioscler Thromb Vasc Biol 17:536–541PubMedCrossRefGoogle Scholar
  26. Peitsch MC (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans 24:274–279PubMedGoogle Scholar
  27. Perschinka H, Mayr M, Millonig G, Mayerl C, van der Zee R, Morrison SG, Morrison RP, Xu Q, Wick G (2003) Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis. Arterioscler Thromb Vasc Biol 23:1060–1065PubMedCrossRefGoogle Scholar
  28. Perschinka H, Wellenzohn B, Parson W, van der Zee R, Willeit J, Kiechl S, Wick G (2007) Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment. Atherosclerosis 194:79–87PubMedCrossRefGoogle Scholar
  29. Pozsonyi E, Gyorgy B, Berki T, Banlaki Z, Buzas E, Rajczy K, Hosso A, Prohaszka Z, Szilagyi A, Cervenak L, Fust G (2009) HLA-association of serum levels of natural antibodies. Mol Immunol 46:1416–1423PubMedCrossRefGoogle Scholar
  30. Prohaszka Z, Duba J, Lakos G, Kiss E, Varga L, Janoskuti L, Csaszar A, Karadi I, Nagy K, Singh M, Romics L, Fust G (1999) Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability. Int Immunol 11:1363–1370PubMedCrossRefGoogle Scholar
  31. Prohaszka Z, Duba J, Horvath L, Csaszar A, Karadi I, Szebeni A, Singh M, Fekete B, Romics L, Fust G (2001) Comparative study on antibodies to human and bacterial 60 kDa heat shock proteins in a large cohort of patients with coronary heart disease and healthy subjects. Eur J Clin Invest 31:285–292PubMedCrossRefGoogle Scholar
  32. Puga Yung GL, Fidler M, Albani E, Spermon N, Teklenburg G, Newbury R, Schechter N, van den Broek T, Prakken B, Billetta R, Dohil R, Albani S (2009) Heat shock protein-derived T-cell epitopes contribute to autoimmune inflammation in pediatric Crohn’s disease. PLoS One 4:e7714PubMedCrossRefGoogle Scholar
  33. Schwede T, Diemand A, Guex N, Peitsch MC (2000) Protein structure computing in the genomic era. Res Microbiol 151:107–112PubMedCrossRefGoogle Scholar
  34. Ulmansky R, Cohen CJ, Szafer F, Moallem E, Fridlender ZG, Kashi Y, Naparstek Y (2002) Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion. J Immunol 168:6463–6469PubMedGoogle Scholar
  35. Uray K, Hudecz F, Fust G, Prohaszka Z (2003) Comparative analysis of linear antibody epitopes on human and mycobacterial 60-kDa heat shock proteins using samples of healthy blood donors. Int Immunol 15:1229–1236PubMedCrossRefGoogle Scholar
  36. van Eden W, Koets A, van Kooten P, Prakken B, van der Zee R (2003) Immunopotentiating heat shock proteins: negotiators between innate danger and control of autoimmunity. Vaccine 21:897–901PubMedCrossRefGoogle Scholar
  37. Varbiro S, Biro A, Cervenak J, Cervenak L, Singh M, Banhidy F, Sebestyen A, Fust G, Prohaszka Z (2010) Human anti-60 kD heat shock protein autoantibodies are characterized by basic features of natural autoantibodies. Acta Physiol Hung 97:1–10PubMedCrossRefGoogle Scholar
  38. Veres A, Fust G, Smieja M, McQueen M, Horvath A, Yi Q, Biro A, Pogue J, Romics L, Karadi I, Singh M, Gnarpe J, Prohaszka Z, Yusuf S (2002) Relationship of anti-60 kDa heat shock protein and anti-cholesterol antibodies to cardiovascular events. Circulation 106:2775–2780PubMedCrossRefGoogle Scholar
  39. Wick G, Xu Q (1999) Atherosclerosis—an autoimmune disease. Exp Gerontol 34:559–566PubMedCrossRefGoogle Scholar
  40. Wu T, Tanguay RM (2006) Antibodies against heat shock proteins in environmental stresses and diseases: friend or foe? Cell Stress Chaperones 11:1–12PubMedCrossRefGoogle Scholar
  41. Wysocki J, Karawajczyk B, Gorski J, Korzeniowski A, Mackiewicz Z, Kupryszewski G, Glosnicka R (2002) Human heat shock protein 60 (409–424) fragment is recognized by serum antibodies of patients with acute coronary syndromes. Cardiovasc Pathol 11:238–243PubMedCrossRefGoogle Scholar
  42. Xu Q, Willeit J, Marosi M, Kleindienst R, Oberhollenzer F, Kiechl S, Stulnig T, Luef G, Wick G (1993) Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis. Lancet 341:255–259PubMedCrossRefGoogle Scholar
  43. Yamaguchi H, Osaki T, Kai M, Taguchi H, Kamiya S (2000) Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori. Infect Immun 68:3448–3454PubMedCrossRefGoogle Scholar
  44. Yi Y, Zhong G, Brunham RC (1993) Continuous B-cell epitopes in Chlamydia trachomatis heat shock protein 60. Infect Immun 61:1117–1120PubMedGoogle Scholar
  45. Zhang X, He M, Cheng L, Chen Y, Zhou L, Zeng H, Pockley AG, Hu FB, Wu T (2008) Elevated heat shock protein 60 levels are associated with higher risk of coronary heart disease in Chinese. Circulation 118:2687–2693PubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2011

Authors and Affiliations

  • George Füst
    • 1
  • Katalin Uray
    • 2
  • László Bene
    • 3
  • Ferenc Hudecz
    • 2
    • 4
  • István Karádi
    • 1
  • Zoltán Prohászka
    • 1
    • 5
  1. 1.3rd Department of Internal MedicineSemmelweis UniversityBudapestHungary
  2. 2.Research Group of Peptide Chemistry, Hungarian Academy of SciencesEötvös L. UniversityBudapestHungary
  3. 3.Hospital of Péterfy SándorBudapestHungary
  4. 4.Institute of ChemistryEötvös L. UniversityBudapestHungary
  5. 5.Research Group of Inflammation Biology and ImmunogenomicsHungarian Academy of Sciences and Semmelweis UniversityBudapestHungary

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