title: A monoclonal antibody (MCPR3-7) interfering with the activity of proteinase 3 by an allosteric mechanism.
abstract: Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1' pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis.
authors.affiliation: From the Comprehensive Pneumology Center, Institute of Lung Biology and Disease (iLBD), University Hospital, Ludwig Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany.
authors.forename: Lisa C
authors.initials: LC
authors.lastname: Hinkofer
authors.forename: Susanne A I
authors.initials: SA
authors.lastname: Seidel
authors.forename: Brice
authors.initials: B
authors.lastname: Korkmaz
authors.forename: Francisco
authors.initials: F
authors.lastname: Silva
authors.forename: Amber M
authors.initials: AM
authors.lastname: Hummel
authors.forename: Dieter
authors.initials: D
authors.lastname: Braun
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Jenne
authors.forename: Ulrich
authors.initials: U
authors.lastname: Specks
mesh_terms: D000495:Allosteric Site; D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015551:Autoimmunity; D002384:Catalysis; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D053146:Myeloblastin / Q000276:immunology*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D014657:Vasculitis / Q000276:immunology
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D053146:Myeloblastin
keywords: Allostery; Antibodies; Autoimmunity; Neutrophil; Protease; Protease Inhibitor; Proteinase 3; Thermophoresis; Zymogen; α1-Proteinase Inhibitor
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