Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment.

Autoimmune reactions to HSP60 are believed to play a key role during development of early atherosclerosis. Due to the high degree of phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for inducing cross-reactivity to self HSP60, which is expressed on the surface of arterial endothelial cells stressed by classical atherosclerosis risk factors. Conformational epitopes recognized by polyclonal anti-mycobacterial HSP60 antibodies from subjects with atherosclerosis were identified using a phage displayed random library of cyclic constrained 7mer peptides. After five rounds of selection, DNA sequencing of strongly binding clones revealed that one peptide motif (CIGSPSTNC) was present in 64% of all clones, and a second motif (CSFHYQNRC) in 14%. Using a newly developed method for structural alignment of small constrained peptides onto a protein surface, we located the motif present in 14% of all clones on the surface of mycobacterial HSP60. The motif present in 64% of all clones was found on the surface of mycobacterial HSP60 as well as in the homologous region of human HSP60, which makes this epitope a promising candidate for further investigations on cross-reactive epitopes involved in early atherogenesis.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA.

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.

Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis.

OBJECTIVE: Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells. METHODS AND RESULTS: We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified. Antisera specific for these cross-reactive epitopes were produced by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease. CONCLUSIONS: Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis.