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The Pseudomonas aeruginosa LasB Metalloproteinase Regulates the Human Urokinase-Type Plasminogen Activator Receptor through Domain-Specific Endoproteolysis

Dominique Leduc,^1,2 Nathalie Beaufort,^1,2, Sophie de Bentzmann,³ Jean-Claude Rousselle,⁴ Abdelkader Namane,⁴ Michel Chignard,^1,2 and Dominique Pidard^1,2*

INSERM, U874, Paris F-75015, France,¹ Unité de Défense Innée et Inflammation, Institut Pasteur, Paris F-75015, France,² CNRS, IBSM-UPR9027, Marseille F-13402, France,³ Plate-Forme de Protéomique, Institut Pasteur, Paris F-75015, France⁴

Received 4 January 2007/ Returned for modification 6 March 2007/ Accepted 2 May 2007

Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala⁸⁴-Val⁸⁵ and Thr⁸⁶-Tyr⁸⁷ (D1-D2) and Gln²⁷⁹-Tyr²⁸⁰ (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa.

Published ahead of print on 21 May 2007.

Editor: J. N. Weiser

Present address: Klinische Forschergruppe der Frauenklinik der Technische Universität München, München, Germany.

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