The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis.
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 Ala(84)-Val(85) and Thr(86)-Tyr(87) (D1-D2) and Gln(279)-Tyr(280) (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.
Leduc
Dominique
D
INSERM, U874, Paris F-75015, France; Unité de Défense Innée et Inflammation, Institut Pasteur, Paris F-75015, France.
Beaufort
Nathalie
N
de Bentzmann
Sophie
S
Rousselle
Jean-Claude
JC
Namane
Abdelkader
A
Chignard
Michel
M
Pidard
Dominique
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology; D005434:Flow Cytometry; D017353:Gene Deletion; D006801:Humans; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000382:microbiology; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D001426:Bacterial Proteins; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa