Molecular analysis of the membrane insertion domain of proteinase 3, the Wegener's autoantigen, in RBL cells: implication for its pathogenic activity

Journal of Leukocyte Biology

Torben Broemstrup³  Arnaud Millet²  Eric Hajjar³  Julie Gabillet²  Chahrazade Kantari²  Paula Pluta³  Nathalie Reuter and¹  Véronique Witko-Sarsat, ² 

[1] Computational Biology Unit, uniBCCS, uniResearch, Bergen, Norway; and Department of Molecular Biology, University of Bergen, Bergen, NorwayINSERM U06, Institut Cochin, Paris, France;  CNRS-UMR 804, Université Paris Descartes, Sorbonne Paris Cité, Paris, France;Computational Biology Unit, uniBCCS, uniResearch, Bergen, Norway;

关键词: neutrophil;    autoimmunity;    vasculitis;    leukemia;    inflammation;   

DOI  :  10.1189/jlb.1210695

学科分类：生理学

来源: Federation of American Societies for Experimental Biology

【 摘 要 】

PR3, also called myeloblastin, is a neutrophil serine protease that promotes myeloid cell proliferation by cleaving the cyclin-dependent kinase inhibitor p21cip1/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.

【 授权许可】

Unknown