RESEARCH - Candidate autoantigens identified by mass spectometry in early RA are chaperones and citrullinated glycolytic enzymes

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Arthritis Research & Therapy 2009, 11:R38doi:10.1186/ar2644

Published: 10 March 2009

Candidate autoantigens identified by mass spectrometry in early

rheumatoid arthritis are chaperones and citrullinated glycolytic

enzymes

Goeb , -L'Otellier , Romain Daveau , Roland

Charlionet , Patrice Fardellone , Xavier Le Loet , Francois Tron ,

e Gilbert and Olivier Vittecoq

Abstract (provisional)

Introduction

The aim of our study was to identify new early rheumatoid arthritis

(RA) autoantibodies.

Methods

Sera obtained from 110 early untreated RA patients (<6 months) were

analyzed by Western blot using HL-60 cell extract, separated on 1- and

2-dimensional gel electrophoresis (1-, 2-DE). Sera from 50 healthy

blood donors and 20 patients with non-RA rheumatisms were used as

controls for 1- and 2-DE respectively. The immunoreactive proteins

were identified by MALDI-TOF mass spectrometric analysis and the

presence of potential sites of citrullination in each of these

proteins was evaluated. FT-ICR Mass Spectrometer was used to verify

experimentally the effect of citrullination upon the mass profile

observed by MALDI-TOF analysis.

Results

The 110 1-DE patterns allowed detection of 10 recurrent immunoreactive

bands of 33, 39, 43, 46, 51, 54, 58, 62, 67 and 70 kDa, which were

further characterized by 2-DE and proteomic analysis. Six proteins

were already-described RA antigens: heterogeneous nuclear

ribonucleoprotein (hnRNP) A2/B1, aldolase, alpha-enolase,

calreticulin, heat shock protein 60 (HSP60) and BiP. Phosphoglycerate

kinase1 (PGK1), stress-induced phosphoprotein 1 and the far upstream

element-binding proteins (FUSE-BP) 1 and 2 were identified as new

antigens. Post-translational protein modifications were analyzed and

potentially deiminated peptides were found on aldolase, alpha-enolase,

PGK1, calreticulin, HSP60 and the FUSE-BPs. We compared the reactivity

of RA sera with citrullinated and non-citrullinated alpha-enolase and

FUSE-BP linear peptides, and showed that antigenicity of the FUSE-BP

peptide was highly dependent on citrullination. Interestingly,

anti-cyclic citrullinated peptide antibody (anti-CCP2) status in RA

serum at inclusion was not correlated to the reactivity directed

against FUSE-BP citrullinated peptide.

Conclusions

Two categories of antigens, enzymes of the glycolytic family and

molecular chaperones are also targeted by the early untreated RA

autoantibody response. For some of them, and notably the FUSE-BPs,

citrullination is involved in the immunological tolerance breakdown

observed earlier in RA patients. Autoantibodies recognizing a

citrullinated peptide from FUSE-BP may enhance the sensibility for RA

of the current available anti-CCP2 test.

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