Biomedical research institute

    Research project 2

    Structural and functional analysis of the autoantigen proteinase 3

    Our aim is to study the mechanisms of neutrophil activation and the role of proteinase 3 in autoimmune necrotizing vasculitis and especially in granulomatosis with polyangitis (GPA, formerly called Wegener’s granulomatosis)

    The pathogenesis of anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides is poorly understood but it is consistent with a pivotal role for neutrophils since they are both effector cells responsible for endothelial damage and targets of autoimmunity.

    Systemic vasculitides include a heterogeneous group of systemic diseases characterized by vascular necrosis and perivascular inflammation in small vessels. Notably, the clinical presentation of these diseases is different and varies according to the localization and the vessel size involved. ANCA-associated vasculitides involve small vessels and comprise microscopic polyangiitis (MPA), Churg-Strauss syndrome and granulomatosis with polyangitis (GPA, formerly called Wegener’s granulomatosis). In both microscopic polyangiitis and Churg-Strauss syndrome, myeloperoxidase is the main target antigen, whereas Wegener’s granulomatosis is strongly associated with ANCA directed against proteinase 3 (PR3).

    In granulomatosis with polyangitis (GPA), neutrophils strongly express membrane-associated proteinase 3, which constitutes a pro-inflammatory factor. Although proteinase 3 appears to play an instrumental role in Wegener’s granulomatosis pathophysiology, its functions in neutrophils are not well understood.


    Our Project is focused on the structural and functional analysis of the autoantigen PR3 to specifically target particular domains to alter its functions.

    Figure 9 : Molecular structure of PR3 showing its catalytic triad and its membrane insertion site with its association with phosphatidylserine on microvesicles.

    Proteinase 3 has the typical fold of chymotrypsin-like serine proteases (Hajjar et al. FEBS J. 2010): two beta-barrels made each of six anti-parallel beta-sheets (purple) and a C-terminal alpha helix (yellow). The active catalytic triad site is located between the two barrels (his71, asp118 and ser203 in green). The substrate (red) is positioned optimally in the active site. PR3 has an hydrophobic patch to insert into the membrane (Hajjar et al Proteins 2008) (shown in yellow and orange)
    (Molecular modeling was performed by the group of Nathalie Reuter, University of Bergen ; Hajjar et al Proteins 2008).

    PR3 could bind to phosphatidylserine expressed on apoptotic cells or on microvesicles to further modulate and disseminate inflammation (Martin et al J Biol Chem, 2016)

    The structural studies on PR3 from the team have provided evidence that PR3 was associated with membrane proteins at the surface of apoptotic neutrophils. PR3 is a  phosphatidylserine–binding protein can modulate the function of proteins involved in the recognition of apoptotic cells including the phospholipidscramblase 1, calreticulin, the C1q fragment of the complement and annexine-A1.

    The presence of PR3 in the proteic platform seems to participate to the pathogenesis of renal vasculitis.

    Figure 10 : PR3 is associated with « eat-me-signal » proteins involved in the clearance and in the immune response related to apoptotic cells.


    Martin KR & Witko-Sarsat V.Proteinase 3: the odd one out that became an autoantigen. J Leukoc Biol. 2017 Sep;102(3):689-698.

    Thieblemont N, Witko-Sarsat V, Ariel A. Regulation of macrophage activation by proteins expressed on apoptotic neutrophils: Subversion towards autoimmunity by proteinase 3. Eur J Clin Invest. 2018 Nov;48 Suppl 2:e12990.

    Thieblemont N, Wright HL, Edwards SW, Witko-Sarsat V. Human neutrophils in auto-immunity. Semin Immunol. 2016 Apr;28(2):159-73.


    Disturbed death pathways in neutrophils from GPA patients

    The functional study of neutrophils from GPA patients in the NEUTRO-VASC clinical study (PH-RC national) was carried out to examine whether measurement of neutrophil activation could be used  as biomarkers in this disease.

    Proteomic analysis of neutrophil cytosol in more than 70 patients with GPA has shown that neutrophils from patients have an intrinsic dysregulation of death pathways and in mechanisms of apoptotic cell recognition thereby explaining the defect in the resolution of inflammation observed in this diseases.

    More importantly, a network of membrane proteins containing the PR3 autoantigen, annexine-A1 and phospholipidscramblase1 was expressed at the surface of apoptotic neutrophils only in GPA patients with a renal involvement suggesting that these proteins were linked to the clinical state and renal disease.

    Figure 11: Individual analysis of neutrophil proteomes from healthy controls  (HC) or GPA patients at basal state or after apoptosis


    Everts-Graber J, Martin KR, Thieblemont N, Mocek J, Roccabianca A, Chafey P, Le Gall M, Tacnet-Delorme P, Reutelingsperger CP, Naccache JM, Bonnotte B, Karras A, Puéchal X, Guillevin L, Terrier B, Frachet P, Perretti M, Mouthon L, Witko-Sarsat V. Proteomic analysis of neutrophils in ANCA-associated vasculitis reveals a dysregulation in proteinase 3-associated proteins such as annexin-A1 involved in apoptotic cell clearance. Kidney Int. 2019 Aug;96(2):397-408.