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International Immunology, Vol. 15, No. 10, pp. 1229-1236, October 2003
© 2003 Japanese Society for Immunology

Comparative analysis of linear antibody epitopes on human and mycobacterial60-kDa heat shock proteins using samples of healthy blood donors

Katalin Uray1, Ferenc Hudecz1, George Füst2 and Zoltán Prohászka2

1 Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, Budapest 112, PO Box 32, 1518 Hungary 2 Third Department of Medicine, Semmelweis Medical University, Budapest, Hungary and Research Group on Metabolism and Atherosclerosis, Hungarian Academy of Sciences, Kutvolgyi st. 4, 1125 Budapest, Hungary

Correspondence to: Z. Prohászka; E-mail: prohoz{at}kut.sote.hu
Transmitting editor: I. Pecht


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Growing evidence suggests that anti-Hsp60 antibodies might be involved in the pathogenesis of different autoimmune diseases. Attempts were reported also on the characterization of epitope specificity of anti-Hsp60 antibodies in different infectious and autoimmune diseases. However, there is a lack of data on the occurrence and epitope specificity of anti-Hsp60 antibodies in the healthy human antibody repertoire. Therefore, the aim of our study was to demonstrate the presence of anti-Hsp60 antibodies and to investigate the epitope specificity of these antibodies using a large set of synthetic 10mer peptides covering regions of Hsp60 with high probability of antigenicity. Here we report the identification of several linear epitopes using serum Ig (IVIG) and sera from healthy subjects by ELISA assay. We have identified two epitopes ‘specific’ for human Hsp60 and two different epitopes ‘specific’ for Hsp65. In addition, six epitopes were cross-reactive in nature, detected on both proteins. The presence of these ‘specific’ epitopes may explain the differences in epitope structure between Hsp60 and Hsp65 observed earlier in patients with cardiovascular disease. The binding of the IVIG preparation to Hsp60 epitopes might indicate that anti-Hsp60 autoantibodies are present in the healthy human natural autoantibody repertoire.

Keywords: human Hsp60, Hsp epitope structure, linear B cell epitopes, Mycobacterium bovis Hsp65, natural autoantibody


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Several studies aimed to determine antibody levels to 60-kDa heat shock proteins (Hsp60). One of the main conclusions of these studies is that Hsp60-specific antibodies are abundant and present even in healthy subjects (1–4). Since Hsp60 is an immunodominant antigen component of different common pathogens, there is a consensus that Hsp60-specific antibodies may be formed in response to such infections. Recent work focused on the characterization of epitope specificity of anti-Hsp60 antibodies in various infectious diseases, including Helicobacter pylori-related gastritis (5), Chlamydia trachomatis related pelvic inflammatory disease (6), periodontitis caused by Porphyromonas gingivalis (7) and scrub typhus caused by Rickettsia tsutsugamushi (8), and in subjects with proven carotid atherosclerosis (9,10). However, these studies did not provide data derived from healthy subjects as a control. These results indicate the presence of multiple, continuous B cell epitopes on Hsp60 and identify some specific epitopes related to infection. Data may also provide certain experimental evidence for infection-induced anti-Hsp60 autoimmunity. However, communications published in the literature also indicate that anti-Hsp60 antibodies might persist independently of infections (11–13).

Recent work (14) has suggested that the 60-kDa Hsp may possess epitopes responsible for pathogenicity, as well as confer resistance to disease induction, e.g. in diabetes mellitus (15) or arthritis (14,16). Important experimental evidence was presented by showing the link between protection for arthritis and the presence of anti-Hsp60 antibodies with certain epitope specificity (14). In addition, Pashov et al. (17) demonstrated the presence of Hsp90-specific antibodies in a large pool of serum Ig from thousands of healthy blood donors (IVIG). Based on these findings, we assume that anti-Hsp60 antibodies in the serum of healthy subjects may represent a portion of the natural autoantibody repertoire, whose formation might be in connection with common infections acquired throughout the life. We reported earlier that anti-Hsp60 IgG antibodies recognized only human Hsp60 in coronary artery disease patients and that the binding could not be inhibited by Hsp65 in a competitive experiment (18). Recent observations in patients with inflammatory bowel disease (IBD) show that although the levels of anti-Hsp65 antibodies were decreased in IBD patients as compared to healthy controls, levels of anti-Hsp60 antibodies were almost identical to those of control subjects (19). These findings indicate different regulation and perhaps different roles of Hsp60- and Hsp65-specific antibodies.

The aim of the present study was to demonstrate anti-Hsp60 autoantibodies in samples of healthy subjects and to investigate the epitope specificity of the Hsp60-specific antibody repertoire using a large set of synthetic 10mer peptides covering regions of Hsp60 with high probability of antigenicity. Our additional objective was to identify epitopes ‘specific’ either for Hsp60 or Hsp65 that might be potentially related to the above-mentioned differences between Hsp60 and Hsp65 epitope structures. However, our study design (comparison of only two members, one bacterial and one mammalian, of the Hsp60 family) might be misleading, since it is impossible to investigate all of the common bacterial or mammalian members of the Hsp60 family. Therefore, in the case of epitopes where sharp differences were observed, we denote our results as ‘specific’. Here, we report on the identification of several linear epitopes by using IVIG and sera from healthy subjects. We found epitopes characteristic for the human or bacterial Hsp and also several epitopes which are cross-reactive in nature.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Prediction of antigenic sites
For the prediction of ß-turns, we used the algorithm of Prevelige and Fasman (20). For the hydrophobicity calculations of protein segments, we used the method of Eisenberg et al. (21). In both cases windows of 7 amino acids were used. Segments with a high probability of ß-turn secondary structure and a low probability of hydrophobicity were considered for selection. We have also obtained three-dimensional models of Hsp60 and Hsp65 proteins from the Swiss-Model Automated Protein Modeling Server (http://www.embl-heidelberg.de/predictprotein/) (22–27). The models were based on the homology in primary protein sequence between human and Escherichia coli GroEL (Hsp60 homologue), and between Mycobacterium bovis Hsp65 and GroEL. For this, the tertiary structure of E. coli GroEL (Brookhaven Protein Data Bank no. 1AON) (28) was utilized. In addition, we also considered published data in the literature (6,8,9) and our pilot experiments for the selection of the regions of Hsp60 to be covered by synthetic peptides.

Peptide synthesis
Decapeptides overlapping by 5 amino acid residues were synthesized on ß-alanyl-glycine functionalized polyethylene pins on two blocks (Mimotopes) with Fmoc/tBu chemistry according to Geysen’s method (29). We used tBu (Thr, Ser, Tyr), OtBu (Asp, Glu), Acm (Cys), Pmc (Arg) and Boc (His, Lys) as side-chain-protecting groups. The Fmoc {alpha}-amino-protecting group was removed with 20% piperidine:DMF (v/v). The coupling was performed with DIC/HOBt methodology and monitored with bromophenol blue added to the coupling mixture (30). The peptides were acetylated at the N-terminus and then the side-chain-protecting groups were removed with TFA:EDT:anisole 38:1:1 (v/v/v), but the unprotected peptides remained covalently attached to the pins. Two control peptides were also prepared: PLAQGGGGGG and GLAQGG GGGG. A specific mouse anti-PLAQ antibody was used to check the validity of the synthesis. The synthesis was successful based on strong reactivity of anti-PLAQGGG GGG mAb to the peptide, showing only background reactivity with peptide GLAQGGGGGG (data not shown). Peptide GLAQGGGGGG was also used later as an unrelated, negative control.

Samples
Purified Ig preparation of >6000 blood donors was tested (IVIG, Humaglobin; Human Rt, Hungary) at 20 mg/l. Twelve (four female) different serum samples from healthy blood donors were also used after careful investigation of the medical history of the subjects and physical examination. Only subjects without any chronic or acute infectious disease in good clinical condition were submitted to blood sampling. Their average age was 54.78 (2.93) years [mean (SD)].

Determination of antibodies reacting with peptides derived from Hsp60 or Hsp65
Antibody binding to the Hsp60 or Hsp65 peptides, immobilized on polyethylene pins, was detected by using a modified ELISA. After blocking the non-specific binding sites (PBS/0.5% gelatin), pins were incubated with 150 µl of 1:500 diluted sera in PBS/0.5% gelatine/0.05% Tween 20 for 1 h at room temperature. Binding of anti-Hsp peptide antibodies was determined using rabbit anti-human IgG peroxidase-labeled antibodies (Dako) and H2O2/o-phenylene-diamine (Sigma) detection system. The optical density was measured at {lambda} = 490 nm (reference at {lambda} = 620 nm) and means of duplicates were calculated. Pins were used repeatedly after thorough cleaning by sonication in disruption buffer (PBS, 1% SDS and 0.1% 2-mercaptoethanol). The following peptides were used for normalization of the respective assay runs: Hsp60172–181 and Hsp65146–155 peptides on block ‘A’, and Hsp60479–488 and Hsp65451–460 peptides on block ‘B’. These peptides showed minimum OD values defined as ODmin in most of the runs. OD/ODmin values for each peptide were calculated, where OD corresponds to the peptide in question. This ratio was very similar to the ratio of the OD value of the peptide in question divided by the OD value of the non-related control peptide GLAQGGGGGG.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We studied both human Hsp60 and M. bovis Hsp65 to allow comparison between the epitope structures of these proteins. Human Hsp60 consists of 573 amino acids and M. bovis Hsp65 consists of 539 amino acids—the difference in length is caused by the mitochondrial targeting sequence. The numbering in this paper corresponds to the proteins with the following accession numbers: human Hsp60 P10809 and mycobacterial Hsp65 P06806. There is a significant level of homology between these sequences (47%) according to BLAST analysis (ftp://ncbi.nlm.nih.gov) (31).

Antigenicity prediction and selection of regions for synthesis
The three-dimensional structures of human Hsp60 and M. bovis Hsp65 are not known. Therefore, to localize the potentially antigenic sites on these proteins we have performed secondary structure and hydrophobicity prediction calculations, assuming that antibody epitopes are usually found at or near hydrophilic ß-turn parts of proteins. We have performed Chou–Fasman secondary structure prediction calculations on human Hsp60 and M. bovis Hsp65 proteins for ß-turns and hydrophobicity calculations. Twenty short sequences (2–11 residues) on Hsp60 showed Pß-turn > 1 values where ß-turns occur with high probability and Phydrophobicity < 0 values showing hydrophilic amino acids. With similar criteria we found 13 shorter sequences (2–7 residues) on M. bovis Hsp65. We have also used a three-dimensional model for human Hsp60 and M. bovis Hsp65 based on the crystal structure of E. coli GroEL. The localization of the selected, potentially antigenic regions of human Hsp60 and their homology-based tertiary structure is presented in Figures 1 and 2.



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  		Fig. 1. The ß-turn secondary structure (solid line) and hydrophobicity calculations (broken line) for human Hsp60. Black periods represent sequences where the probability value of ß-turn is >1 and the hydrophobicity value is negative.

 


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  		Fig. 2. Three-dimensional model of human Hsp60 monomer based on the tertiary structure of E. coli Hsp60. The peptides synthesized are marked in black, the sequences marked by the ribbon structure are: (A) human Hsp60 ‘specific’ epitopes, (B) M. bovis Hsp65 ‘specific’ epitopes, and (C) common epitopes between human Hsp60 and M. bovis Hsp65 recognized by healthy human sera. The insert shows the localization of a GroEL monomer in the oligomeric structure of GroEL-GroES machinery consisting of 14 GroEL monomers and an additional ring of seven GroES monomers (‘cap’).

 
Considering experimental data on epitopes of M. bovis and other homologous bacterial Hsp65 proteins (6,8,9) and our pilot experiments (data not shown), we have included the following 10 protein regions into our studies: I: amino acids 52–81, II: amino acids 117–146, III: amino acids 162–186, IV: amino acids 203–262, V: amino acids 303–308, VI: amino acids 368–387, VII: amino acids 394–413, VIII: amino acids 436–455, IX: amino acids 470–509 and X: amino acids 531–550 (according to Hsp60 numbering) (Table 1). We have synthesized decamer peptides corresponding to the selected regions with a 5-amino-acid overlap. To make the comparison between peptides based on the human Hsp60 or M. bovis Hsp65 sequence possible, twin peptides of the human Hsp60 peptides, according to the homologue region on Hsp65, were also synthesized in parallel. A total of 92 decamer peptides (46 and 46 according to Hsp60 and Hsp65 respectively) plus four control peptides (unrelated sequences without homology to any of the Hsp molecules) were synthesized in duplicate.


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  		Table 1. Sequence alignment of the 10 regions selected for synthesis within M. bovis Hsp65 and human Hsp60
 
Binding of serum antibodies to pin-attached peptides
Binding of serum IgG antibodies from healthy individuals to the pin-attached human Hsp60 and M. bovis Hsp65 peptides was measured. Two kinds of samples were tested. The first one was an i.v. Ig preparation (IVIG). It was obtained by purification from the pooled plasma of >6000 healthy blood donors. The second set of samples was collected from individual blood donors (n = 12).

As a negative control we used the unrelated peptide GLAQGGGGGG. No sequential homology exists between this peptide and Hsp60 or Hsp65 proteins. In order to eliminate the plate-to-plate variations we selected two Hsp60 and two Hsp65 peptides, which exhibited the lowest OD values, as internal controls. Using these non-binding compounds we have calculated ODmin as described in Methods.

We first calculated ratios of average OD/ODmin for each peptide, and then the mean and SD of the ratios in all experiments for all plates. The reactivity of the peptides was classified based on the OD/ODmin values representing the binding between of samples to the individual peptides. Four groups of peptides were established by considering OD/ODmin values in the following ranges: high reactivity (+++): mean + 2 SD < OD/ODmin, medium reactivity (++): mean + 1 SD < OD/ODmin <= mean + 2 SD, low reactivity (+): mean + 0.5 SD < OD/ODmin <= mean + 1 SD and no reactivity (–): OD/ODmin <= mean + 0.5 SD. To analyze the binding of the individual sera with these peptides in depth, the proportion of positive sera (ratio above mean + 1 SD, i.e. ++ and +++) in the groups was calculated. Table 2 shows the 10 regions and all 10mer peptides derived from human Hsp60 and M. bovis Hsp65 proteins. Peptides from the respective proteins are listed as pairs and the level of sequential homology is indicated by the underlined amino acids. It should be noted that this value could differ significantly (0–80%).


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  		Table 2. Reactivity of IVIG and serum samples derived from healthy subjects with synthetic peptides derived from human Hsp60 and M. bovis Hsp65 proteins
 
First, we analyzed the binding of IVIG to peptides in the 10 regions of Hsp60. These data show marked reactivity towards peptides in the I–III and VIII regions. In these regions some peptides possess at least score ++. Almost similar binding profiles could be detected with individual sera. At least one peptide in these regions bound to serum antibodies with a score ++ (medium reactivity). The peptides recognized with at least medium reactivity by both IVIG and individual sera are Hsp6062–71, Hsp6067–76, Hsp60132–141, Hsp60167–176 and Hsp60441–450. In addition, the recognition of peptides Hsp6052–61, Hsp60218–227, Hsp60228–237 and Hsp60436–445 by individual sera was also pronounced. In the case of Hsp6057–66, Hsp6072–81, Hsp60117–126 and Hsp60137–146, we observed low reactivity with individual sera, but medium reactivity with IVIG. Peptide Hsp60394–403 showed only low reactivity, but with both IVIG and individual sera.

Next, we analyzed the binding of IVIG to peptides in the 10 Hsp65 regions. In regions II, III, IV, VIII and X we observed reactivity towards some peptides using the same criteria as above. Individual sera recognized peptides from regions I, II, IV, VII and X. In three regions (II, IV and X), both IVIG and the individual sera recognized peptides Hsp6591–100, Hsp65191–200 and Hsp65507–516. The recognition by individual serum samples was also pronounced with peptides Hsp6526–36, Hsp6531–40, Hsp6536–45, Hsp65111–120, Hsp65196–205 and Hsp65366–375. We also observed low reactivity (+) towards M. bovis peptide Hsp65276–285 in region V by both IVIG and individual sera. Peptide Hsp65502–511 bound to IVIG very strongly, but had no reaction with individual sera.

Next we compared the antibody binding of the homologous human Hsp60 and M. bovis Hsp65 peptides. We observed some epitopes ‘specific’ to either human Hsp60 or M. bovis Hsp65. In region I, peptides Hsp6067–76 and Hsp6072–81, and in region II, peptide Hsp60132–141, are recognized by sera, but their M. bovis homologues are inactive. On the other hand, the M. bovis peptide Hsp65507–516 from region X and (to a lesser degree) peptides Hsp65502–511 and Hsp65276–285 were recognized, but their human homologues were not.

We also observed cross-reactive recognition in three cases: Hsp6057–66 versus Hsp6531–40, Hsp60117–126 versus Hsp6591–100 and Hsp60394–403 versus Hsp65366–375. Some degree of similar binding was also documented with Hsp6052–61 versus Hsp6526–35, Hsp6062–71 versus Hsp6536–45, Hsp60137–146 versus Hsp65111–120, Hsp60218–227 versus Hsp65191–200 and Hsp60441–450 versus Hsp65413–422. These peptides show 40–70% homology.

We next analyzed the location of human or M. bovis ‘specific’ epitope peptides within the respective proteins according to the homology and prediction-based tertiary structure of Hsp60 or Hsp65. This structure is based on the E. coli GroEL structure, which is built up of 14 identical proteins in two heptamers. The consecutive human Hsp6067–76 and Hsp6072–81 have a turn structure between a sheet and a helix in the protein; this turn is on the surface of the monomer Hsp60. However, taking into consideration the fact that the functional Hsp molecule occurs in oligomers, this part of the molecule is probably on the contact surface between the monomeric units and not on the surface. Hsp60132–141 peptide also contains a turn/loop structure between two helices; its E. coli GroEL homologue is on the surface of heptameric, but on the contact surface of the 14mer protein. M. bovis ‘specific’ epitope Hsp65276–285 is a loop on the contact surface of monomeric proteins. M. bovis peptides Hsp65502–511 and Hsp65507–516, containing a helical structure, are also on the inner or contact surface of the oligomers.

Furthermore, we have analyzed the position of cross-reactive epitope peptides. Peptide Hsp6057–66 and its M. bovis homologue Hsp6531–40 of region I consist of loop and ß-sheet structures on the surface of the monomer Hsp60, but probably on the contact surface in oligomers. This is consistent with the observation of Ulmansky et al. (14) who described the same region as a surface localized epitope on Hsp65. The common epitope peptide in region II (Hsp60117–126 and Hsp6591–100) is mainly helical; it contains hindered and surface parts as well, but the latter ones are probably on the contact surface. The third common epitope (Hsp60394–403 and Hsp65366–375) with lower reactivity consists of an accessible loop structure located between an {alpha}-helix and a ß-sheet, they are on the surface of the polymeric Hsp. We have also analyzed the position of the cross-reactive epitopes with a lower degree of similarity. Epitopes in region I (Hsp6052–61, Hsp6062–71 and their M. bovis homologues) all contain loop/turn structures on the surface of monomers, but are hindered in oligomeric proteins. In region II, the cross-reactive epitope Hsp60137–146 versus Hsp65111–120 is also on the contact surface, but it is helical. Epitope Hsp60167–176 versus Hsp65141–150 in region III is helical and on the surface of the oligomer. The common epitope Hsp60218–227 versus Hsp65191–200 in region IV consists of sheet and loop structures; it is partly on the contact surface, partly hindered. Peptides Hsp60441–450 versus Hsp65413–422 in region VIII are of a loop and helix structure, and are on the outer surface of the oligomer.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
There is perfect agreement in the literature on the common presence of antibodies to Hsp60 in healthy humans (1–4). Several studies aimed at the investigation of continuous B cell epitopes of the Hsp60 family in various infectious diseases (5–8); however, epitope analysis of the antibody repertoire of healthy humans has not been reported so far. Therefore, we aimed to determine the specificity of anti-Hsp60 antibodies in healthy human blood donors. Hsp60 is an immunodominant protein of different common (e.g. H. pylori, C. pneumoniae) and specific (R. tsutsugamushi, P. gingivalis) microbes causing acute or chronic diseases. The human homologue protein shows high-level (47%) similarity to the microbial protein (32), giving rise to infection-induced autoimmunity (33). Therefore, our study was designed in a comparative manner to allow investigation of cross-reactivity. In addition, we intended to analyze the linear B cell epitope background of differences in epitope structure between Hsp60 and Hsp65 (18). In this paper we show the presence of two epitopes solely recognized on human Hsp60 and two other epitopes characteristic for Hsp65. These data are in accordance with our earlier observation (19), and further strengthen our conception on the different regulation of anti-Hsp60 and anti-Hsp65 antibodies and their diverse associations with certain diseases. However, most of the linear epitopes investigated were cross-reactive in nature, i.e. recognized on both proteins.

We observed antibody binding to peptides in a total of eight regions. The antibody binding was cross-reactive in nature in three (region I/amino acids 52–81, region II/amino acids 117–146 and region VII/amino acids 394–413, according to the Hsp60 numbering) of these regions, i.e. peptides in the same localization on Hsp60 and Hsp65 were both recognized. Weaker cross-reactivity was also observed in regions III, IV and VIII. According to the literature, parts of regions I, II and IV are recognized in different infectious diseases (6–8). Based on our results, we assume that these regions of the Hsp60 are candidate sites to induce cross-reactive antibodies. Supporting this assumption is the observation of Metzler et al. (9) who localized three sites on Hsp65 where antibody binding occurs in samples of patients with carotid atherosclerosis. Two out of the three regions overlapped or mapped near to the cross-reactive epitopes (region II/amino acids 117–131 and region IV/amino acids 218–237) detected in our study. The C-terminal of the epitope defined by Metzler et al. also overlaps by 3 amino acids with the human ‘specific’ Hsp60132–141 peptide. In the third region we did not observe cross-reactivity; only the M. bovis peptides were recognized according to Hsp65 region X/amino acids 502–521. In a recent paper by the same group (10), eight cross-reactive epitopes of human Hsp60 were described using a series of overlapping peptides and high-titer anti-Hsp65 antibodies. Two out of the eight epitopes described in that study and also by Metzler et al. (9) were detected in our current experiments, and were found cross-reactive. Six epitopes were either not predicted by us (n = 4) or no antibody binding was observed (n = 2). Hajeer et al. in 1992 (34) described antigenic sites on Hsp65 recognized by mAb; the IIH9 mAb described there has an epitope nearly identical to the cross-reacting Hsp65111–120 peptide.

The cross-reactive regions described in the present study and in the literature (region I, II, IV and VIII) were also recognized by the IVIG preparation; the fourth cross-reactive IVIG epitope in region VIII overlaps with the so-called p277 peptide (15). Most importantly, all of our IVIG epitopes on Hsp65 were recently described by Ulmansky et al. to elicit protective antibody reaction against adjuvant arthritis in rat (14). Taken together, the published data and our results provide further evidence for the possible induction of natural, protecting autoantibodies, including anti-Hsp60 antibodies, through common infections. The best reflection of the natural autoantibody repertoire is the IVIG preparation and several epitopes recognized by these antibodies on Hsp60 or Hsp65 are cross-reactive in nature. Furthermore, these anti-Hsp antibodies may play a significant role in the regulation of autoimmune processes as presented by their ability to protect against adjuvant arthritis (14,16) or diabetes mellitus (15). The presence of natural antibodies to certain epitopes may actually inhibit T cell responses to them, whereas the lack of antibodies enables the T cells to respond to these epitopes. In addition, the presence of antibodies to a potential autoantigen may result in the skewing of the local cytokine profile toward an anti-inflammatory response. Experimental evidence for this mechanism was elegantly shown recently (14) in the rat adjuvant arthritis model. Furthermore, the human Hsp60167–176 epitope showed the strongest reactivity with IVIG, indicating an important role for this region in human Hsp60-specific autoimmunity.

The two M. bovis ‘specific’ epitopes are located on the inner surface of Hsp (being on the surface of monomer Hsp only). The two human Hsp60 ‘specific’ epitopes are at very much exposed parts of the monomer, but are probably parts of the contact surfaces between monomers. According to prediction and three-dimensional protein modeling, most of the cross-reactive epitopes are located on the contact surface of the Hsp60/65 molecule, with the exceptions of Hsp60167–176, Hsp60394–403 and Hsp60441–450.

In contrast to cross-reactive epitopes, several regions were described where IVIG and/or individual serum samples reacted only with the bacterial peptide of the twin peptides (e.g. Hsp65276–285 and Hsp65502–511). It is important to note that the presence of antibodies against all of these epitopes was described earlier in one or more infectious diseases where epitope analysis of 60-kDa Hsp was performed (6–8). Therefore, these epitopes are candidate sites for vaccine development eliciting pathogen-specific humoral immunity.

According to our knowledge this is the first attempt to characterize the epitope specificity of Hsp60 antibodies from healthy subjects. We are aware that there are several limitations of our study. One of the difficulties lies in the lack of generally accepted consensus methods for the definition of a ‘positive antibody reaction’ to peptides. Considering that this might significantly influence the interpretation of data, we have defined a scale of reactivity. Although we considered positive antibody reactivity above the mean +1 SD level, in biological terms this interpretation may be misleading. Furthermore, our method is limited to the identification of linear antibody epitopes and not capable of detecting topographic antigenic sites.

In conclusion, we were able to describe linear B cell epitopes characteristic for either human Hsp60 or bacterial Hsp65. Most importantly, our data provide further evidence for the occurrence of anti-Hsp60 antibodies in the healthy human natural autoantibody repertoire as detected by the IVIG preparation. The finding that all of the cross-reactive epitopes were recognized by IVIG strengthens the hypothesis that the healthy human protective autoantibody repertoire may be induced by common infections.


   Acknowledgements
 
These studies were supported by grants from the Ministry of Education (FKFP 0101/97, FKFP 0138/01 and MEC-01431/00), Ministry of Welfare (ETT 248/2001) and from the National AIDS Committee (NABKP-4/98).


   Abbreviations
 
Hsp—heat shock protein

IBD—inflammatory bowel disease

IVIG—i.v. Ig preparation


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Xu, Q., Kiechl, S., Mayr, M., Metzler, B., Egger, G., Oberhollenzer, F., Willeit, J. and Wick, G. 1999. Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis. Clinical significance determined in a follow-up study. Circulation 100:1169.[Abstract/Free Full Text]
  2. Veres, A., Fust, G., Smieja, M., McQueen, M., Horvath, A., Yi, Q., Biro, A., Pogue, J., Romics, L., Karadi, I., Singh, M., Gnarpe, J., Prohászka, Z. and Yusuf, S. 2002. Heart Outcomes Prevention Evaluation (HOPE) Study Investigators. Relationship of anti-60 kDa heat shock protein and anti-cholesterol antibodies to cardiovascular events. Circulation 106:2775.[Abstract/Free Full Text]
  3. Rea, I. M., McNerlan, S. and Pockley, A. G. 2001. Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp. Gerontol. 36:341.[CrossRef][Web of Science][Medline]
  4. Zhu, J., Quyyumi, A. A., Rott, D., Csako, Gy., Wu, H., Halcox, J. and Epstein, S. E. 2001. Antibodies to human heat shock protein 60 are associated with the presence and severity of coronary artery disease. Evidence for an autoimmune component of atherosclerosis. Circulation 103:1071.[Abstract/Free Full Text]
  5. Yamaguchi, H., Osaki, T., Kai, M., Taguchi, H. and Kamiya, S. 2000. Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori. Infect. Immun. 68:3448.[Abstract/Free Full Text]
  6. Yi, Y., Zhomg, G. and Brunham, R. 1993. Continuous B-cell epitopes in Chlamydia trachomatis heat shock protein 60. Infect. Immun. 61:1117.[Abstract/Free Full Text]
  7. Maeda, H., Miyamoto, M., Kokeguchi, S., Kono, T., Nishimura, F., Takashiba, S. and Murayama, Y. 2000. Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis. FEMS Immunol. Med. Microbiol. 28:219.[CrossRef][Web of Science][Medline]
  8. Lachumanan, R., Devi, S., Cheong, Y. M., Rodda, S. and Pang, T. 1993. Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi. Infect. Immun. 61:4527.[Abstract/Free Full Text]
  9. Metzler, B., Schett, G., Kleindienst, R., van der Zee, R., Ottenhoff, T., Hajeer, A., Bernstein, R., Xu, Q. and Wick, G. 1997. Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 17:536.[Abstract/Free Full Text]
  10. Perschinka, H., Mayr, M., Millonig, G., Mayerl, C., van der Zee, R., Morrison, S. G., Morrison, R. P., Xu, Q. and 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.[Abstract/Free Full Text]
  11. Prohászka, Z., Duba, J., Horváth, L., Császár, A., Karádi, I. and Szebeni, A. 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.[CrossRef][Web of Science][Medline]
  12. Burian, K., Kis, Z., Virok, D., Endrész, V., Prohászka, Z., Duba, J., Berencsi, K., Boda, K., Romics, L., Füst, G. and Gönczöl, 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.[Abstract/Free Full Text]
  13. Veres, A., Szamosi, T., Ablonczy, M., Szamosi, T., Jr, Singh, M., Karádi, I., Romics, L., Füst, G. and Prohászka, Z. 2002. Complement activating antibodies against the human 60 kDa heat shock protein as a new independent family risk factor of coronary heart disease. Eur. J. Clin. Invest. 32:405.[CrossRef][Web of Science][Medline]
  14. Ulmansky, R., Cohen, C. J., Szafer, F., Moallem, E., Fridlender, Z. G., Kashi, Y. and 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.[Abstract/Free Full Text]
  15. Elias, D. and Cohen, I. R. 1995. Treatment of autoimmune diabetes and insulitis in NOD mice with heat shock protein 60 peptide p277. Diabetes 44:1132.[Abstract]
  16. Yang, X. D., Gasser, J. and Feige, U. 1990. Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein. Clin. Exp. Immunol. 81:189.[Web of Science][Medline]
  17. Pashov, A., Kenderov, A., Kyurkchiev, S., Kehayov, I., Hristova, S., Lacroix-Desmazes, S., Giltiay, N., Varamballi, S., Kazatchkine, M. D. and Kaveri, S. V. 2002. Autoantibodies to heat shock protein 90 in the human natural antibody repertoire. Int. Immunol. 14:453.[Abstract/Free Full Text]
  18. Prohászka, Z., Duba, J., Lakos, G., Kiss, E., Varga, L., Jánoskuti, L., Császár, A., Karadi, I., Nagy, K., Singh, M., Romics, L. and Füst, G. 1999. Antibodies against human hsp60 and mycobacterial hsp65 differ in their antigen specificity and complement activating ability. Int. Immunol. 11:1363.[Abstract/Free Full Text]
  19. Bene, L., Füst, G., Huszti, Z., Hernádi, Zs., Fekete, B., Mészáros, M., Veres, A., Kovács, Á., Miklós, K., Singh, M., Romics, L. and Prohászka, 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.[CrossRef][Web of Science][Medline]
  20. Prevelige, P. and Fasman, G. D. 1990. Chou–Fasman prediction of the secondary structure of proteins. In Fasman, G. D., ed., Prediction of Protein Structure and the Principles of Protein Conformation, p. 391. Plenum Press, New York.
  21. Eisenberg, D., Wesson, M. and Wilcox, W. 1990. Hydrophobic moments as tools for analyzing protein sequences and structures. In Fasman, G. D., ed., Prediction of Protein Structure and the Principles of Protein Conformation, p. 635. Plenum Press, New York.
  22. Peitsch, M. C. 1995. Protein modeling by e-mail. Biotechnology 13:658.[CrossRef]
  23. Peitsch, M. C. 1996. Promod and Swiss-Model: Internet-based tools for automated comparative protein modeling. Biochem. Soc. Trans 24:274.[Web of Science][Medline]
  24. Peitsch, M. C. and Guex, N. 1997. Large-scale comparative protein modeling. In Wilkins, M. R., Williams, K. L., Appel, R. D and Hochstrasser, D. F., eds, Proteome Research: New Frontiers in Functional Genomics, p. 177. Springer, New York.
  25. Peitsch, M. C. and Guex, N. 1997. Swiss-Model and the Swiss-PDBViewer: an environment for comparative protein modeling. Electrophoresis 18:2714.[CrossRef][Web of Science][Medline]
  26. Guex, N., Diemand, A. P. and Peitsch, M. C. 1999. Protein modeling for all. Trends Biochem. Sci. 24:364.[CrossRef][Web of Science][Medline]
  27. Schwede, T., Diemand, A., Guex, N. and Peitsch, M. C. 2000. Protein structure computing in the genomic era. Res. Microbiol. 151:107.[Medline]
  28. Xu, Z., Horwich, A. L. and Sigler, P. B. 1997. The crystal structure of the asymetic GroEl–GroES (ADP)7 chaperonin complex. Nature 388:741.[CrossRef][Medline]
  29. Geysen, H. M., Meloen, R. H. and Barteling, S. J. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl Acad. Sci. USA 81:3998.[Abstract/Free Full Text]
  30. Krchnák, V., Vágner, J., Safár, P. and Lebl, M. 1988. Noninvasive continuous monitoring of solid-phase peptide synthesis by acid–base indicator. Collection Czechoslovak Chem. Commun. 53:2542.
  31. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.[Abstract/Free Full Text]
  32. Singh, B. and Gupta, R. S. 1992. Expression of human 60-kD heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies. DNA Cell Biol. 11:489.[Web of Science][Medline]
  33. Jones, D. B., Coulson, A. F. and Duff, G. W. 1993. Sequence homologies between Hsp60 and autoantigens. Immunol. Today 14:115.[CrossRef][Web of Science][Medline]
  34. Hajeer, A. H., Worthington, J., Morgan, K. and Bernstein, R. M. (1992) Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning. Clin. Exp. Immunol. 89:115.

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K. Udvarnoki, L. Cervenak, K. Uray, F. Hudecz, I. Kacskovics, R. Spallek, M. Singh, G. Fust, and Z. Prohaszka
Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins
Clin. Vaccine Immunol., April 1, 2007; 14(4): 335 - 341.
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