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.

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

“…In addition, unlike other neutrophil granule proteins, PR3 is coexternalized with phosphatidylserine (PS) during neutrophil apoptosis because of its association with phospholipid scramblase 1 and impaired macrophage phagocytosis (5). We recently reported that PR3, which is a peripheral membrane protein, has a hydrophobic patch essential for its membrane anchorage and for its proinflammatory activity (6). Accordingly, a hydrophobic patch-deficient PR3 mutant was not externalized on apoptosis and did not impair phagocytosis (6).…”

“…We recently reported that PR3, which is a peripheral membrane protein, has a hydrophobic patch essential for its membrane anchorage and for its proinflammatory activity (6). Accordingly, a hydrophobic patch-deficient PR3 mutant was not externalized on apoptosis and did not impair phagocytosis (6).…”

“…Phagocytosis of apoptotic RBL cells by human monocyte-derived macrophages 7 d, the culture medium was removed and human monocyte-derived macrophages were incubated with TAMRA-labeled apoptotic RBL cells (at a ratio of 1:1) in 2 ml Iscove's DMEM for 2 h, as described previously (6), to quantify the cytokines secreted in culture supernatants with the RayBio human inflammation Ab array or the Quantikine ELISA kit (R&D Systems) according to the manufacturer's instructions. For the phagocytosis of apoptotic neutrophils from healthy donors and vasculitis patients, macrophages were incubated with a mouse monoclonal anti-LRP Ab (America Diagnostica) for 1 h, then with TAMRA-labeled apoptotic neutrophils (at a ratio of 10:1) in 2 ml Iscove's DMEM for 2 h. After phagocytosis, the macrophages were washed twice with PBS/EDTA (2 mM), and phagocytosis was analyzed using flow cytometry (21).…”

“…Cells were selected on their neomycin resistance and cloned. Apoptosis was induced by 16-h incubation at 37˚C with 2 mg/ml gliotoxin (Sigma), as described previously (6). Degranulation was induced with 2 mM A23187 calcium ionophore and was evaluated by measuring b-hexosaminidase activity in the degranulation supernatant, using the chromogenic substrate p-nitrophenyl-N-acetyl-b-D-glucosaminide (Sigma), as described previously (18).…”

“…Recombinant human PR3 or the membrane-anchoragedeficient PR3 mutant, PR34H4A, was expressed in stably transfected RBL cells as described previously (6,19). RBL/CD177 and RBL/ CD177-PR3 were obtained by transfecting RBL or RBL/PR3 cells with pcDNA3-neomycin containing CD177 cDNA (GeneCopoeia) using the AMAXA system as described previously (6).…”