Pseudomonas aeruginosa Protease IV Enzyme Assays and Comparison to Other Pseudomonas Proteases

Caballero, Armando R.; Moreau, Judy M.; Engel, Lee S.; Marquart, Mary E.; Hill, James M.; O’Callaghan, Richard J.

doi:10.1006/abio.2001.4999

Cited by 129 publications

(122 citation statements)

References 44 publications

(21 reference statements)

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“…Elastin Congo Red Assay-The elastolytic activity of each enzyme was tested by a modification of the procedures of Rust et al (35,36). 10 mg of Congo Red elastin (Sigma) were suspended in 0.9 ml of 50 mM sodium acetate buffer (pH 5.5) containing 2.5 mM DTT and 2.5 mM EDTA (for cysteine protease), or 50 mM Tris-HCl (pH 7.2) with or without 5 mM CaCl 2 (for metalloproteinases and serine proteases, respectively).…”

Section: Methodsmentioning

confidence: 99%

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Yasuda

Greenbaum

et al. 2004

Journal of Biological Chemistry

174

138

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Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.Atherosclerosis is characterized by arterial intimal enlargement and subsequent lipid deposition leading to the formation of blood stream obstructing plaques. The infiltration of monocyte-derived macrophages (MDMs) 1 and smooth muscle cells (SMCs) into the intima of the inflamed artery contributes to the formation of atherosclerotic lesions. Atherosclerotic lesions resident MDMs and SMCs produce a large number of extracellular matrix-degrading enzymes (1), such as cysteine proteases (2-5) and matrix metalloproteinases (MMPs) (6 -8).Elastin and collagen are the two major extracellular matrix components that provide elasticity and tensile strength to the arterial wall. The destruction of elastin and collagen causes a weakening and rupture of blood vessels (9, 10). MMPs, serine, and cysteine proteases have been identified as major elastolytic proteases in arteries (11, 12). Among cysteine proteases, cathepsins S and K have been considered as the most potent elastolytic activities with cathepsin K exhibiting a slightly higher activity than cathepsin S (13, 14). However, cathepsin K-deficient human macrophages derived from patients with pycnodysostosis were shown to retain their high cysteine proteasedependent elastolytic activities (15). This finding implied that additional cathepsins may contribute to the overall elastolytic activity in human macrophages.We and others (16 -18) have identified and partially characterized a novel human cysteine protease closely related to cathepsin L that was named cathepsin V (also known as cathepsin L2). Cathepsin V shares 80% protein s...

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Section: Methodsmentioning

confidence: 99%

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Yasuda

Greenbaum

et al. 2004

Journal of Biological Chemistry

174

138

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“…We examined the proteolytic activities of lecA and lecB mutants by growing them on casein-milk agar plates. In P. aeruginosa, caseinolytic activity is due mainly to the actions of LasB, alkaline protease, and protease IV (6). After 36 h, the wildtype strain, the lecA mutant, and the complemented lecB mutant produced a proteolytic zone, while a very weak zone was produced by the lecB mutant (Fig.…”

Section: Fig 1 Proteomic Profile Of the Outer Membranes Of P Aerugmentioning

confidence: 98%

“…4B), suggesting that the reduced caseinolytic activity is due not to a LasB defect but to either alkaline protease or protease IV. Alkaline protease is a metalloprotease whose activity is inhibited by EDTA (6). Therefore, to eliminate alkaline protease, we took advantage of the specific ability of protease IV to cleave lactoferrin in the presence of EDTA (31).…”

Section: Fig 1 Proteomic Profile Of the Outer Membranes Of P Aerugmentioning

confidence: 99%

Pseudomonas aeruginosa LecB Is Involved in Pilus Biogenesis and Protease IV Activity but Not in Adhesion to Respiratory Mucins

2006

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Pseudomonas aeruginosa expresses two lectins which are implicated in adhesion and biofilm formation. In this study, we demonstrate that P. aeruginosa LecB is involved in pilus biogenesis and proteolytic activity. Moreover, neither lectin was involved in adhesion to human tracheobronchial mucin. We infer that some of the ascribed functions are secondary effects on other systems rather than effects of the lectins themselves.

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“…LasA protease can also nick elastin at certain Gly-Gly sequences (9,10), an activity that increases the susceptibility of elastin to other proteases and contributes to the elastinolytic potential of P. aeruginosa (9,11,12). The fourth endopeptidase secreted by P. aeruginosa (lysine-specific endopeptidase; protease IV) cleaves peptide bonds on the carboxyl side of lysine residues in peptides and proteins and can act on a number of host proteins including complement components, IgG, and fibrinogen (13)(14)(15). LasD, a secreted protein first described as a second staphylolytic protease of P. aeruginosa (16), is apparently not a protease but may function as a chitin-binding protein (17).…”

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confidence: 99%

A Secreted Aminopeptidase of Pseudomonas aeruginosa

Cahan¹,

Axelrad²,

Safrin³

et al. 2001

Journal of Biological Chemistry

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Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was ϳ56 kDa; hence, it was designated AP 56 . Heating (70°C) of the partially purified aminopeptidase preparations led to the conversion of AP 56 to a ϳ28-kDa protein (AP 28 ) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP 28 was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine-or thiol-proteases, suggesting that AP 28 is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP 56 and AP 28 were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39 -58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, ϳ35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29 -32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

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Pseudomonas aeruginosa Protease IV Enzyme Assays and Comparison to Other Pseudomonas Proteases

Cited by 129 publications

References 44 publications

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Pseudomonas aeruginosa LecB Is Involved in Pilus Biogenesis and Protease IV Activity but Not in Adhesion to Respiratory Mucins

A Secreted Aminopeptidase of Pseudomonas aeruginosa

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