Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.
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