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Cleavage of Annexin‐A1 by Proteinase 3: a possible cause of Wegener’s vasculitis?
Author(s) -
Vong Linda,
D’Acquisto Fulvio,
Flower Roderick J,
WitkoSarsat Veronique,
Perretti Mauro
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a645-d
Subject(s) - annexin , in vitro , cleavage (geology) , chemistry , microbiology and biotechnology , transfection , cytosol , annexin a2 , endogeny , mutant , biochemistry , enzyme , biology , gene , paleontology , fracture (geology)
Annexin‐A1 (Anx‐A1) is an endogenous anti‐inflammatory mediator with potent anti‐migratory effects on polymorphonuclear leukocytes (PMN). Several studies have shown Anx‐A1 N‐terminal cleavage (between Val‐36 and Ser‐37) accompanies an inflammatory stimulus, and may serve to alter its biological function in PMN. In this study we set up an in vitro assay to measure the Anx‐A1‐specific proteolytic activity in human PMN. To this aim, an Anx‐A1 construct (MFA1) was generated tagged with c‐Myc and FLAG epitopes at the N‐ and C‐terminal, respectively. This construct was transfected into HEK 293T cells, and the recombinant protein purified from the cell extracts for use as a substrate. Our results demonstrate the proteolytic activity towards MFA1 resides exclusively in the PMN‐derived membrane, but not cytosolic fractions. This was inhibited with the addition of MeOSuc‐Ala‐Ala‐Pro‐Val chloromethyl ketone, a PR3 inhibitor. Addition of purified human PR3 or HMC1 cell extracts where PR3 is overexpressed (HMC1/PR3) also resulted in MFA1 cleavage. In contrast, MFA1‐specific proteolytic activity was absent in extracts of HMC1 where the inactive PR3 mutant (HMC1/S207A) is overexpressed. These results suggest PR3 to be responsible for the cleavage of Anx‐A1 in PMN. Relevantly, this phenomenon may contribute to the development of Wegener’s granulomatosis, a form of vasculitis characterized by an exaggerated PMN activation and accumulation in the vascular wall. This work is supported by Bart’s and The London Special Trustees (JRB XMSR).