Journal of Biological Chemistry
Volume 293, Issue 32, 10 August 2018, Pages 12415-12428
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Enzymology
Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

https://doi.org/10.1074/jbc.RA118.001922Get rights and content
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Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon–Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5–4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

protease
protease inhibitor
neutrophil
aminopeptidase
antigen
autoimmune disease
genetic disease
proteinase 3
cathepsin C
granulomatosis with polyangiitis
Papillon-Lefèvre syndrome

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This work was supported by the “Ministère de l'Enseignement Supérieur et de la Recherche,” the “Région Centre-Val de Loire” (Project BPCO-Lyse). This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement 668036 (RELENT). The authors declare that they have no conflicts of interest with the contents of this article. Responsibility for the information and views set out in this article lies entirely with the authors.

This article contains Figs. S1–S6.

4

U. Specks, personal communication.

5

S. Seren, S. Dallet-Choisy, F. Gauthier, S. Marchand-Adam, and B. Korkmaz, unpublished results.

1

These authors contributed equally to this work.

3

The abbreviations used are:

    GPA

    granulomatosis with polyangiitis

    α1PI

    α-1-proteinase inhibitor

    Ab

    antibody

    ABZ

    ortho-aminobenzoic acid

    ANCA

    anti-neutrophil cytoplasmic autoantibodies

    Bt

    biotin

    CatC

    CatL, and CatS, cathepsin C, L, and S, respectively

    CG

    cathepsin G

    ECL

    enhanced chemiluminescence

    EDDnp

    N-(2,4-dinitrophenyl)ethylenediamine

    HNE

    human neutrophil elastase

    HSC

    hematopoietic stem cell(s)

    NSP

    neutrophil serine protease

    PLS

    Papillon–Lefèvre syndrome

    PR3

    proteinase 3

    PR3m

    membrane-bound PR3

    WBC

    white blood cell(s)

    NET

    neutrophil extracellular trap

    IcatC

    CatC inhibitor

    PE

    phycoerythrin

    APC

    allophycocyanin.