Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

Kreij, Arno de; Venema, Gerard; Burg, Bertus van den

doi:10.1074/jbc.m003889200

Cited by 46 publications

(30 citation statements)

References 45 publications

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“…Unlike LL-37, HKH20 is highly resistant to various bacterial proteases (not shown), likely because of the absence of hydrophobic residues in HKH20. Notably, the P. aeruginosa elastase, E. faecalis gelatinase, or the 50-kDa metalloproteinase of P. mirabilis belong to the M4 peptidase family (thermolysin family) and have similar specificities requiring hydrophobic amino acids (Leu, Ile, Phe) at the P1Ј position (48,49). Taken together, from a therapeutical standpoint, our results suggest that strategies based on utilizing endogenous and protease-resistant AMPs with high therapeutic indexes could be highly rewarding.…”

Section: Discussionmentioning

confidence: 75%

Domain 5 of High Molecular Weight Kininogen Is Antibacterial

Nordahl

Rydengård

Mörgelin

et al. 2005

Journal of Biological Chemistry

102

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Antimicrobial peptides are important effectors of the innate immune system. These peptides belong to a multifunctional group of molecules that apart from their antibacterial activities also interact with mammalian cells and glycosaminoglycans and control chemotaxis, apoptosis, and angiogenesis. Here we demonstrate a novel antimicrobial activity of the heparin-binding and cell-binding domain 5 of high molecular weight kininogen. Antimicrobial epitopes of domain 5 were characterized by analysis of overlapping peptides. A peptide, HKH20 (His 479 -His 498 ), efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Enterococcus faecalis. Fluorescence microscopy and electron microscopy demonstrated that HKH20 binds to and induces breaks in bacterial membranes. Furthermore, no discernible hemolysis or membrane-permeabilizing effects on eukaryotic cells were noted. Proteolytic degradation of high molecular weight kininogen by neutrophil-derived proteases as well as the metalloproteinase elastase from P. aeruginosa yielded fragments comprising HKH20 epitopes, indicating that kininogen-derived antibacterial peptides are released during proteolysis.The innate immune system, based on antimicrobial peptides (AMP), 2 provides a rapid and nonspecific response against potentially invasive pathogenic microorganisms. AMPs, first isolated from human leukocyte extracts by Zeya and Spitznagel in 1963 (1), were subsequently discovered in invertebrates (2) and cold-blooded vertebrates (3). At present, over 880 different AMPs have been identified in eukaryotes. During recent years it has become increasingly evident that many antimicrobial peptides are multifunctional (4, 5). They are found to mediate chemotaxis (defensins, LL-37) (6 -8), apoptosis (lactoferricin, LL-37) (9 -11), and angiogenesis (PR-39, LL-37) (12, 13). Conversely, molecules previously not considered as AMPs, including proinflammatory and chemotactic chemokines (14), neuropeptides (15-19), and peptide hormones (16,20), have recently been found to exert antibacterial activities. The proinflammatory, chemotactic, and anaphylatoxic peptide C3a, generated during activation of the complement system, displays potent antibacterial effects (21). Many AMPs, by virtue of their cationicity and amphipathicity, also interact with heparin (22, 23).High molecular weight kininogen (HMWK) is a multifunctional 120-kDa glycoprotein found in plasma (ϳ80 g/ml) (24) and in ␣-granules of platelets (25). The protein is composed of six domains, each having different properties and specific ligands (24). Domains D1-D3 have a cystatin-like structure, and the two latter domains serve as specific inhibitors of cysteine proteinases such as cathepsins and calpains. The D4 domain contains the bradykinin sequence, which is released by plasma kallikreins during contact activation (24, 26). Biologically active kinins can also be generated by the cooperative action of mast cell tryptase and neutrophil elastase (27). Thus, similar to complement degrad...

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Section: Discussionmentioning

confidence: 75%

Domain 5 of High Molecular Weight Kininogen Is Antibacterial

Nordahl

Rydengård

Mörgelin

et al. 2005

Journal of Biological Chemistry

102

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“…Phosphoramidon is a peptide that binds to a specific sequence in the thermolysin active-site region. Sequence differences in this region have been correlated with reduced phosphoramidon sensitivity (13).…”

Section: Discussionmentioning

confidence: 99%

“…ZmpB has a preproenzyme structure that is typical of the M4 family (http://merops.sanger.ac.uk/) of metalloproteases, with conservation of functional regions such as the HExxH active-site motif as well as other conserved amino acids downstream of the active site, including Gxxx ExxxD. De Kreij et al (13) found that there was a great deal of sequence variability among various thermolysin-like proteases but that as long as a small subset of amino acids was conserved (e.g., active site residues and zinc ligands), the group was extremely homogeneous in terms of catalysis. B. cenocepacia ZmpB is inhibited by EDTA and 1,10-phenanthroline, indicating that it is a metalloprotease.…”

Section: Discussionmentioning

confidence: 99%

Burkholderia cenocepacia ZmpB Is a Broad-Specificity Zinc Metalloprotease Involved in Virulence

et al. 2006

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In previous studies we characterized the Burkholderia cenocepacia ZmpA zinc metalloprotease. In this study, we determined that B. cenocepacia has an additional metalloprotease, which we designated ZmpB. The zmpB gene is present in the same species as zmpA and was detected in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria, and B. pyrrocinia but was absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The zmpB gene was expressed, and ZmpB was purified from Escherichia coli by using the pPROEXHTa His 6 Tag expression system. ZmpB has a predicted preproenzyme structure typical of thermolysin-like proteases and is distantly related to Bacillus cereus bacillolysin. ZmpB was expressed as a 63-kDa preproenzyme precursor that was autocatalytically cleaved into mature ZmpB (35 kDa) and a 27-kDa prepropeptide. EDTA, 1,10-phenanthroline, and Zn 2؉ cations inhibited ZmpB enzyme activity, indicating that it is a metalloprotease. ZmpB had proteolytic activity against ␣-1 proteinase inhibitor, ␣ 2 -macrogobulin, type IV collagen, fibronectin, lactoferrin, transferrin, and immunoglobulins. B. cenocepacia zmpB and zmpA zmpB mutants had no proteolytic activity against casein and were less virulent in a rat agar bead chronic infection model, indicating that zmpB is involved in B. cenocepacia virulence. Expression of zmpB was regulated by both the CepIR and CciIR quorumsensing systems.

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“…Thermolysin-like proteases that are more phylogenically related to thermolysin are sensitive to phosphoramidon. Phosphoramidon was specifically designed for thermolysin, and sequence differences in thermolysin-like proteases correlate with decreased phosphoramidon sensitivity (11).…”

Section: B Cenocepaciamentioning

confidence: 99%

Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease

2005

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Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His 6 tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His 6 -pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H 465 with G 465 or A 465 , E 377 with A 377 or D 377 , or H 380 with P 380 or A 380 . Mutagenesis of H 465 , E 377 , or H 380 resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H 380 was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil ␣-1 proteinase inhibitor, ␣ 2 -macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.

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Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

Cited by 46 publications

References 45 publications

Domain 5 of High Molecular Weight Kininogen Is Antibacterial

Domain 5 of High Molecular Weight Kininogen Is Antibacterial

Burkholderia cenocepacia ZmpB Is a Broad-Specificity Zinc Metalloprotease Involved in Virulence

Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease

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