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Abstract

Objective

To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti–endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD.

Methods

The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization−time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis).

Results

Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be α-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant α-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human α-enolase. BD patient sera positive for anti–α-enolase did not react with human γ-enolase. On dot-blotting, reactivity to human α-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant α-enolase IgM antibody by ELISA.

Conclusion

The α-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to α-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, α-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.