title: Molecular analysis of the membrane insertion domain of proteinase 3, the Wegener's autoantigen, in RBL cells: implication for its pathogenic activity.
abstract: PR3, also called myeloblastin, is a neutrophil serine protease that promotes myeloid cell proliferation by cleaving the cyclin-dependent kinase inhibitor p21(cip1/waf1). In addition, it is the target of ANCA in GPA, a necrotizing vasculitis. Anti-PR3 ANCA binding to membrane-expressed PR3 triggers neutrophil activation, potentiating vascular inflammation. This study performed in RBL cells identifies the structural motifs of PR3 membrane anchorage and examines its impact on PR3 proinflammatory and proliferative functions. With the use of MD simulations and mutagenesis, we demonstrate that the mutations of four hydrophobic (F180, F181, L228, F229) or four basic (R193, R194, K195, R227) amino acids abrogated PR3 membrane anchorage. The hydrophobic patch-deficient PR3 mutant (PR34H4A) was still able to cleave the synthetic substrate Boc-Ala-Pro-Val in cell lysates. However, in contrast to WT PR3, PR34H4A was not expressed at the plasma membrane after degranulation and failed to cleave extracellular fibronectin, was not externalized after apoptosis and did not impair macrophage phagocytosis of apoptotic cells, did not promote myeloid cell proliferation and failed to cleave p21/waf1. PR3 membrane insertion appears to be pivotal for its proinflammatory activities, such as extracellular proteolysis and impairment of apoptotic cell clearance, but also for myeloid cell proliferation. Targeting membrane-associated PR3 might constitute a novel, anti-inflammatory therapeutic strategy in inflammatory disease especially in vasculitis, but this approach has to be validated in mature neutrophils.
authors.affiliation: INSERM U1016, Institut Cochin, Paris, France.
authors.forename: Chahrazade
authors.initials: C
authors.lastname: Kantari
authors.forename: Arnaud
authors.initials: A
authors.lastname: Millet
authors.forename: Julie
authors.initials: J
authors.lastname: Gabillet
authors.forename: Eric
authors.initials: E
authors.lastname: Hajjar
authors.forename: Torben
authors.initials: T
authors.lastname: Broemstrup
authors.forename: Paula
authors.initials: P
authors.lastname: Pluta
authors.forename: Nathalie
authors.initials: N
authors.lastname: Reuter
authors.forename: Véronique
authors.initials: V
authors.lastname: Witko-Sarsat
mesh_terms: D000818:Animals; D017209:Apoptosis; D001491:Basophils / Q000201:enzymology / Q000276:immunology* / Q000648:ultrastructure; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D049109:Cell Proliferation; D007249:Inflammation; D009154:Mutation; D053146:Myeloblastin / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018375:Neutrophil Activation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D051381:Rats
chemical_list: D053146:Myeloblastin