Katalin Uray

Antibodies against different epitopes of heat-shock protein 60 in children with type 1 diabetes mellitus

The aim of this study was to investigate the amounts and epitope specificity of antibodies against heat shock protein 60 (hsp60) in the sera of type 1 diabetic and healthy children. Antibodies specific for peptide p277 of human hsp60 and of M. bovis as well as for human hsp60, M. bovis hsp65 proteins were measured by ELISA. Other autoantibodies (islet cell antibodies, glutamate decarboxylase antibodies and IA-2 antibodies) were also determined. A total number of 83 serum samples from children with type 1 diabetes mellitus and 81 samples of control children were investigated. Epitope scanning of the hsp60 for linear antibody epitopes was carried out using synthetic peptides attached to pins. The antibody levels specific for peptide p277 of human- and of M. bovis origin were significantly (human: P=0.0002, M. bovis: P=0.0044) higher in the diabetic children group than in the healthy children. We could not find significant difference in the antibody levels to whole, recombinant hsp proteins among the examined groups of children. Antibodies to two epitope regions on hsp60 (AA394-413 and AA435-454) were detected in high titres in sera of children with diabetes mellitus. The first region similar to the sequence found in glutamate decarboxylase, whereas the second one overlaps with p277 epitope to a large extent. Presence of antibodies to certain epitopes of hsp60 (AA394-413-glutamic acid decarboxylase-like epitope; AA435-454-p277-like epitope) in diabetic children may reflect their possible role in the autoimmune diabetogenic process of the early diabetes.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3β-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

Receptors specific for the Fc part of IgG (FcγR) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory FcγRmay result in autoimmunity. Thus, the modulation of IgG‐FcγR interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to FcγRII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P234 and S298 were synthesized and used in binding and functional experiments. Binding of the peptides to FcγR was assayed in vitro and ex vivo, and peptides foundto interact were functionally tested. The shortest effective peptide was T256–P271, which bound to soluble recombinant FcγRIIb with Kd=6×106 M–1. The biotinylated peptides R255–P271 and T256–P271 complexed by avidin exhibited functional activity; they induced FcγRIIb‐mediated inhibition of the BCR‐triggered Ca2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF‐α and IL‐6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the FcγRII‐binding part of IgG1.

Antibodies against C-reactive protein cross-react with 60-kilodalton heat shock proteins.

ABSTRACT C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.

Synthesis and antibody recognition of mucin 1 (MUC1)-α-conotoxin chimera

We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The Pro‐Asp‐Thr‐Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a β‐turn conformation. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, which is stabilized by two disulphide bridges in positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, Arg9‐His‐Tyr‐Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4‐(2′,4′‐dimethoxyphenyl‐aminomethyl)phenoxy) (Rink) resin and either 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3–Cys13 or Cys2–Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES‐MS) and amino acid analysis. The CD spectra of the bicyclic MUC1‐α‐[Tyr1]‐conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1‐α‐[Tyr1]‐conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1‐α‐[Tyr1]‐conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization. Copyright

Peptide epitopes: identification and structural modifications of synthetic antigens

The application of biologicals like monoclonal antibodies (MoAbs), their fragments, fusion proteins, synthetic epitope peptides, conjugates, nanostructures in fundamental biomedicinal research as well as in early diagnosis and/or efficient and specific targeted therapy including vaccination in various diseases (e.g. cancer, infectious and autoimmune diseases) is on the rise. In this chapter an outline is provided to summarise recent advances, together with the brief references to early attempts, on strategies and approaches developed for identification of protein epitopes, for the manipulation of their structure to achieve optimal immune recognition. By the help of selected examples from the last few years we also survey the application of these discoveries in understanding the details of interactions between proteins, peptides, in the development of epitope based immunodiagnostics or therapeutics.