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1.  Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases 
Biological Chemistry  2010;391(6):705-716.
Elafin is a 6 kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases (neutrophil elastase (NE) and proteinase 3) with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), were able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaved elafin at the amino-terminal Lys6-Gly7 peptide bond resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidences that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.
PMCID: PMC3396147  PMID: 20370321
Antimicrobials; antiproteases; cleavage; microbial; proteases; transglutaminase
2.  COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta 
Human mutation  2011;32(6):598-609.
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
PMCID: PMC3103631  PMID: 21344539
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
3.  The Collagen V Homotrimer [α1(V)]3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems 
Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly.
PMCID: PMC2896673  PMID: 20625483
4.  Role of the Netrin-like Domain of Procollagen C-Proteinase Enhancer-1 in the Control of Metalloproteinase Activity* 
The Journal of Biological Chemistry  2010;285(21):15950-15959.
The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.
PMCID: PMC2871463  PMID: 20207734
ADAM; ADAMTS; Collagen; Extracellular Matrix; Heparin; Matrix Metalloproteinase; Tolloid Proteinase
5.  Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease▿  
Journal of Bacteriology  2007;189(11):3960-3968.
LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.
PMCID: PMC1913401  PMID: 17351039
6.  Pseudomonas aeruginosa LasA Protease in Treatment of Experimental Staphylococcal Keratitis 
LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 × 106 (MSSA) and 5 × 105 (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 × 106 and 5 × 105 CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.
PMCID: PMC400535  PMID: 15105121
7.  Correlation of Virulence and Collagenolytic Activity in Entamoeba histolytica 
Infection and Immunity  1983;39(2):528-531.
The levels of collagenolytic activity of strains HM-1:1 MSS (HM-1), (HM-1), 200-NIH, and HK-9 of Entamoeba histolytica were compared. Collagen degradation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conditioned media as well as extracts of the highly virulent strain HM-1 effectively degraded native type I collagen. Significantly lower activity was found in the analogous fractions of strains 200-NIH and HK-9, which are not as virulent. The collagenolytic activity of strain HM-1 was associated with the isolated plasma membrane fraction and could be eluted from the membranes by buffers of high ionic strength, indicating that it is not an integral membrane protein. Unlike the vertebrate and the clostridial collagenases, the collagenolytic activity of E. histolytica HM-1 was enhanced in presence of dithiothreitol and was inhibited by N-ethylmaleimide. The correlation between virulence of the individual strains and their collagenolytic activity suggests that collagenase might play a role in pathogenesis of amoebiasis. The localization of the enzyme on the plasma membrane and its presence in the extracellular medium favor this view.
PMCID: PMC347982  PMID: 6299942
8.  In Vitro Inhibition of Pseudomonas aeruginosa Elastase by Metal-Chelating Peptide Derivatives 
Infection and Immunity  1982;38(2):716-723.
Pseudomonas aeruginosa elastase is a zinc metalloendopeptidase, probably responsible for the tissue destruction observed during infections with this organism. The elastase of a virulent Pseudomonas aeruginosa strain (Habs serotype 1) was isolated and found to have a molecular weight of 35,000; it readily degraded elastin and cartilage proteoglycans. A series of amino acid and peptide derivatives containing the metal-chelating moieties hydroxamate, phosphoryl, or thiol were synthesized and tested as potential inhibitors of the enzyme. Inhibition constants (Kis) for the compounds were determined with the chromophoric substrate furylacryloyl-glycyl-l-leucyl-l-alanine. The hydroxamic acid derivatives of benzyloxycarbonyl-glycine, benzyloxycarbonyl-l-leucine and benzyloxycarbonyl-l-phenylalanine had inhibition constants in the range of 11 to 28 μM. The 2-mercaptoacetyl derivatives of l-leucyl-d-phenylalanine and l-leucyl-l-phenylalanine had Ki values of 34 and 1.5 μM, respectively, demonstrating the stereospecificity of the inhibition. The most potent inhibitors tested were 2- mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylala-nine (Ki = 0.2 μM). Similar compounds lacking the metal-chelating moiety were about 3 orders of magnitude poorer inhibitors. When the inhibition of the enzyme activity towards azocasein, elastin, or cartilage was examined, inhibitor concentrations approximately 50-fold higher than the respective Kis were required to obtain 60 to 90% inhibition. Virtually complete inhibition was achieved with these substrates at inhibitor concentrations 500-fold higher than the respective Kis (0.1 to 14 mM). Although, 2-mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylalanine exhibited the same affinity to the enzyme, the latter was inferior in inhibiting cartilage proteoglycan degradation. 2-Mercaptoacetyl-l-phenylalanyl-l-leucine represents a class of potent elastase inhibitors that might prove useful in the management of P. aeruginosa infections.
PMCID: PMC347797  PMID: 6815099

Results 1-8 (8)