title: Discrimination and variable impact of ANCA binding to different surface epitopes on proteinase 3, the Wegener's autoantigen.
abstract: Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener's granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1-5/Mh1-5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system.
authors.affiliation: Thoracic Diseases Research Unit, Stabile Bldg. 8-56, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
authors.forename: Francisco
authors.initials: F
authors.lastname: Silva
authors.forename: Amber M
authors.initials: AM
authors.lastname: Hummel
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Jenne
authors.forename: Ulrich
authors.initials: U
authors.lastname: Specks
mesh_terms: D000818:Animals; D056648:Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002469:Cell Separation; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D014890:Granulomatosis with Polyangiitis / Q000276:immunology* / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D051379:Mice; D053146:Myeloblastin / Q000037:antagonists & inhibitors* / Q000276:immunology; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D053146:Myeloblastin
references.citation: J Am Soc Nephrol. 2006 May;17(5):1235-42
references.pmid: 16624929
references.citation: Arthritis Rheum. 1990 Aug;33(8):1101-7
references.pmid: 2202308
references.citation: Arthritis Rheum. 2008 May;58(5):1546-51
references.pmid: 18438818
references.citation: Exp Nephrol. 1995 Jul-Aug;3(4):249-56
references.pmid: 8590038
references.citation: Biochimie. 2008 Feb;90(2):227-42
references.pmid: 18021746
references.citation: Nature. 1990 Aug 9;346(6284):520
references.pmid: 2377228
references.citation: Clin Exp Immunol. 2001 Jan;123(1):170-7
references.pmid: 11168015
references.citation: J Immunol Methods. 2005 Dec 20;307(1-2):62-72
references.pmid: 16242707
references.citation: Clin Exp Rheumatol. 2004;22(6 Suppl 36):S7-12
references.pmid: 15675127
references.citation: Kidney Int. 2003 Feb;63(2):756-60
references.pmid: 12631144
references.citation: J Autoimmun. 2008 Feb-Mar;30(1-2):29-36
references.pmid: 18162369
references.citation: FEBS Lett. 2005 Oct 10;579(24):5305-12
references.pmid: 16182289
references.citation: J Clin Invest. 1989 Nov;84(5):1577-87
references.pmid: 2681270
references.citation: J Leukoc Biol. 2001 Feb;69(2):177-90
references.pmid: 11272267
references.citation: Clin Exp Immunol. 1998 Nov;114(2):320-6
references.pmid: 9822293
references.citation: Clin Immunol. 2002 May;103(2):196-203
references.pmid: 12027425
references.citation: Arthritis Rheum. 1993 Mar;36(3):365-71
references.pmid: 8452581
references.citation: Arthritis Res. 2000;2(4):263-7
references.pmid: 11094439
references.citation: Clin Exp Immunol. 2002 Sep;129(3):562-70
references.pmid: 12197900
references.citation: Ann Intern Med. 2007 Nov 6;147(9):611-9
references.pmid: 17975183
references.citation: Arthritis Rheum. 1994 Feb;37(2):187-92
references.pmid: 8129773
references.citation: Clin Exp Immunol. 1995 Oct;102(1):112-9
references.pmid: 7554377
references.citation: Am J Pathol. 1990 Nov;137(5):1113-20
references.pmid: 2240162
references.citation: Kidney Int. 1995 Jun;47(6):1528-36
references.pmid: 7643521
references.citation: Ann Intern Med. 1989 Jul 1;111(1):28-40
references.pmid: 2660645
references.citation: Am J Med. 2007 Jul;120(7):643.e9-14
references.pmid: 17602941
references.citation: Clin Exp Rheumatol. 2009 Jan-Feb;27(1 Suppl 52):S45-52
references.pmid: 19646346
references.citation: J Immunol. 2010 Jul 1;185(1):387-99
references.pmid: 20530264
references.citation: Clin Exp Immunol. 2004 Jan;135(1):164-72
references.pmid: 14678279
references.citation: Clin Exp Immunol. 1993 Sep;93(3):405-10
references.pmid: 8370167
references.citation: Clin Exp Immunol. 1992 Dec;90(3):409-14
references.pmid: 1458677
references.citation: J Biol Chem. 2008 Dec 19;283(51):35976-82
references.pmid: 18854317
references.citation: Ann Rheum Dis. 2007 Dec;66(12):1679-82
references.pmid: 17644551
references.citation: J Immunol. 1994 May 1;152(9):4722-37
references.pmid: 8157982
references.citation: J Mol Biol. 1996 Aug 16;261(2):267-78
references.pmid: 8757293
references.citation: Blood. 2004 Sep 1;104(5):1411-8
references.pmid: 15150076
references.citation: Proteins. 2008 Jun;71(4):1655-69
references.pmid: 18076025
references.citation: Kidney Int. 1998 Mar;53(3):743-53
references.pmid: 9507222
references.citation: FEBS Lett. 1997 May 19;408(2):187-90
references.pmid: 9187364
references.citation: J Immunol Methods. 1998 Feb 1;211(1-2):111-23
references.pmid: 9617836
references.citation: Clin Exp Immunol. 1995 Mar;99(3):486-92
references.pmid: 7882573
references.citation: FEBS Lett. 1996 Jul 29;390(3):265-70
references.pmid: 8706874
references.citation: Clin Exp Immunol. 1999 Dec;118(3):487-96
references.pmid: 10594572
references.citation: Curr Opin Rheumatol. 2002 Jan;14(1):15-22
references.pmid: 11790991
references.citation: Arthritis Rheum. 2000 Sep;43(9):2025-33
references.pmid: 11014352
references.citation: J Immunol Methods. 2005 Oct 20;305(1):33-8
references.pmid: 16125193
references.citation: Clin Exp Immunol. 1997 Aug;109(2):286-95
references.pmid: 9276524
references.citation: Ann Intern Med. 1997 Jun 1;126(11):866-73
references.pmid: 9163287
references.citation: Clin Exp Immunol. 2006 Oct;146(1):15-20
references.pmid: 16968393
references.citation: J Immunol. 1992 Aug 15;149(4):1409-15
references.pmid: 1380042