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Journal of Biochemistry Advance Access published online on December 11, 2006
Journal of Biochemistry, doi:10.1093/jb/mvm008
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© 2006 The Japanese Biochemical Society

Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.

Ulrich Specks*, David N. Fass{dagger}, Javier D. Finkielman*, Amber M. Hummel*, Margaret A. Viss*, Robert D. Litwiller{dagger} and Cari J. McDonald*

Thoracic Disease Research Unit(*)and Hematology Research Unit ({dagger}), Mayo Clinic and Foundation, Rochester, MN 55905, USA

Corresponding author: Ulrich Specks MD, Thoracic Diseases Research Unit, Stabile Bldg. 8-56, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905. Phone: 507 284 2301. Fax: 507 284 4521, e-mail: specks.ulrich{at}mayo.edu


  Abstract

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.

Key Words: Neutrophils, Protein Processing, Post-Translational, Serine Endopeptidases, Wegener's Granulomatosis


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