Human Hsp40 proteins, DNAJA1 and DNAJA2, as potential targets of the immune response triggered by bacterial DnaJ in rheumatoid arthritis

Abstract

Hsp40 proteins of bacterial and human origin are suspected to be involved in the pathogenesis of rheumatoid arthritis (RA). It has been shown that sera of RA patients contain increased levels of antibodies directed to bacterial and human Hsp40s. The aim of this work was to explore immunological similarities between the bacterial (DnaJ) and human (DNAJA1 and DNAJA2) Hsp40 proteins in relation to their possible involvement in the RA. Using polyclonal antibodies directed against a full-length DnaJ or its domains, against DNAJA1 and DNAJA2, as well as monoclonal anti-DnaJ antibodies, we found immunological similarities between the bacterial and human Hsp40s. Both ELISA and Western blotting showed that these similarities were not restricted to the conserved J domains but were also present in the C-terminal variable regions. We also found a positive correlation between the levels of the anti-DnaJ and anti-DNAJA1 antibodies in the sera of RA patients. This finding supports the molecular mimicry hypothesis that human Hsp40 could be the targets of antibodies originally directed against bacterial DnaJ in RA.

Fig. 1

Cross-reactions between anti-DNAJ(Hsp40) polyclonal antibodies and DNAJ(Hsp40) proteins tested by Western blotting. The DnaJ, N-DnaJ (DnaJΔ107-375), C-DnaJ (DnaJΔ1-199), DNAJA1, and DNAJA2 recombinant proteins were overproduced in E. coli cells, purified, and resolved by SDS-PAGE. Of each Hsp40 protein, 2.5 μg has been loaded onto the gel (a). The polyclonal antibodies indicated above the panels were used in Western blotting as the primary antibodies (b–f)

Fig. 2

Cross-reactions between anti-DNAJ(Hsp40) polyclonal antibodies and DNAJ(Hsp40) proteins tested by ELISA. The reactivity of polyclonal anti-DnaJ (a), anti-N-DnaJ (b), anti-C-DnaJ (c), anti-DNAJA1 (d), and anti-DNAJA2 (e) with purified non-denatured DNAJ antigens was tested as described in “Materials and methods.” Data obtained for the antigens at 40 μg per milliliter are presented as the mean ± SEM of three independent experiments and expressed as a percentage of control (i.e., reactivity with the antigen used to raise a given antibody; indicated as the gray bar)

Fig. 3

Reactivity of anti-DnaJ monoclonal antibodies (mAbs) with human DNAJA proteins. Schematic outline of the DnaJ linear structure and localization of the epitopes recognized by the mAbs are presented in a. The reactivity of six mAbs (b–g) with purified non-denatured DNAJ(Hsp40) antigens was assayed by ELISA test as described in “Materials and methods.” Data obtained for the antigens at 40 μg per milliliter are presented as the mean ± SEM of three independent experiments and expressed as a percentage of the reactivity with DnaJ control (gray bar)