Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity.

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.

Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays.

In this paper we describe the use of a commercial surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray. By monitoring the angle shift of the SPR-dip using a continuous angle-scanning mode instead of monitoring the change in reflectivity at a fixed angle, a linear relationship with respect to the mass density change on the surface will remain over a wide dynamic angle range of 8 degrees. Peptides (2.4 kDa) and proteins (150 kDa) were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. By using a fluorescently labeled antibody, SPR results can be confirmed by means of fluorescence microscopy after completion of a SPR experiment. SPR imaging in angle-scanning operation provides sensitive, accurate, and label-free detection of analyte binding on microarrays containing different molecular weight ligands.

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance.

INTRODUCTION: Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. METHODS: Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays. RESULTS: In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the ß-chain. CONCLUSIONS: A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging.

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.