Anti-neutrophil cytoplasm antibodies in Wegener’s granulomatosis recognize an elastinolytic enzyme (1).
Anti-neutrophil cytoplasm antibodies (ANCA) is closely associated with Wegener’s granulomatosis. It is important to understand the autoantigen of ANCA in the pathogenesis of the disease. ACPA are directed against an elastinolytic neutral serine proteinase, proteinase 3, which is the third neutral serine proteinase of human neutrophils.
ACPA antigens were purified by an affinity column with bound IgG from an ACPA positive serum. The antigens were analyzed by SDS-PAGE and immunoblotting.
The affinity purified antigens were applied to SDS-PAGE under reducing condition or non-reducing conditions, followed by immunoblotting.
Under reducing condition SDS-PAGE revealed two bands (26 and 28 kDa), and under non-reducing condition SDS-PAGE revealed one band (38 kDa).
The amino acid sequence of the N terminus of the antigen was obtained as Ile-Val-Gly-Gly-His-Glu-Ala-Gln-Pro-His-Ile-Arg-Pro-Ile-Tyr-Met-Ala. The sequence has homology with human neutrophils, elastase, cathepsin G, and proteinase 3.
The enzyme activity was assayed using alpha-naphthyl acetate, a substrate being hydrolyzed by many proteinases. The antigen showed enzyme activity of 60% of elastase, and 130% of cathepsin G. However, the antigen failed to cleave elastase and cathepsin G specific substrates. The enzyme activity was not inhibited by pepstatin, N-ethylmaleimide, EDTA, soybean trypsin inhibitor but by PMSF. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile, the antigen can be concluded as proteinase 3.