Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA.
BACKGROUND
Autoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes.


METHODS
This study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA.


RESULTS
The rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA.


CONCLUSIONS
This expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely.
Capizzi
Stephen A
SA
Thoracic Diseases Research Unit and Hematology Research Unit, Mayo Graduate School of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Viss
Margaret A
MA
Hummel
Amber M
AM
Fass
David N
DN
Specks
Ulrich
U
D000595:Amino Acid Sequence / Q000235:genetics; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology*; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D053146:Myeloblastin; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000276:immunology*
D019268:Antibodies, Antineutrophil Cytoplasmic; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin