Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles

Abstract

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3

Regulation of macrophage activation by proteins expressed on apoptotic neutrophils: Subversion towards autoimmunity by proteinase 3

“…88,90 Because PR3 expression was increased at the surface of neutrophils associated with apoptotic markers, such as phosphatidylserine (annexin V pos 7AAD neg ) in GPA patients, it has been postulated that PR3-induced through apoptosis was deleterious and control of apoptosis was defective in GPA patients. 89 Various reports validate the hypothesis that proteins already known as PR3 partners (CRT, AnxA1, PLSCR1) and involved in apoptosis and apoptotic cell clearance would be dysregulated in neutrophils from GPA patients. 84,88 It has also been recently established that PR3 impaired C1q enhancement of apoptotic cell uptake enlighting a novel potential axis in autoimmune diseases characterized by a defect in apoptotic cell clearance and in the resolution of inflammation.…”

“…This results in hampered signalling by the CRT receptor, lipoprotein receptor‐related protein (LRP) 1, by limiting the production of anti‐inflammatory cytokines of macrophages and enhancing inflammatory cytokines, such as the IL‐1β, IL6 and TNF‐α . Similarly, PR3 binds PS and this interaction depends on the hydrophobic patch responsible for membrane anchorage . It is still unknown whether PR3 modulates NET formation or soluble NET would modify PR3 expression.…”

“…As a result, macrophages exposed to apoptotic neutrophils that express PR3 expressed increased levels of the M1 marker inducible NO synthase (iNOS) and secreted higher levels of inflammatory cytokines and chemokines . Because PR3 expression was increased at the surface of neutrophils associated with apoptotic markers, such as phosphatidylserine (annexin V pos 7AAD neg ) in GPA patients, it has been postulated that PR3‐induced through apoptosis was deleterious and control of apoptosis was defective in GPA patients . Various reports validate the hypothesis that proteins already known as PR3 partners (CRT, AnxA1, PLSCR1) and involved in apoptosis and apoptotic cell clearance would be dysregulated in neutrophils from GPA patients .…”

“…84 Similarly, PR3 binds PS and this interaction depends on the hydrophobic patch responsible for membrane anchorage. 89 It is still unknown whether PR3 modulates NET formation or soluble NET would modify PR3 expression. The proinflammatory responses of PR3 require both its enzymatic activity and its membrane anchorage suggesting that recognition of PR3 at the surface of apoptotic cells involves multimolecular interactions.…”

Expanding Neutrophil Horizons: New Concepts in Inflammation

“…Microvesicles are surrounded by a membrane containing high levels of phosphatidylserine, a lipid that is normally located inside the cell. Interestingly, using a unique hydrophobic patch, neutrophil-derived PR3 can bind to phosphatidylserine, a major component of microvesicles, thereby affecting both their production and their function to ultimately promote inflammation [70]. Conversely, microvesicles can have potent anti-inflammatory effects.…”