Chemical Synthesis and Biological Activity of The Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalis and Its Analogs

An 11-residue peptide lactone, termed the gelatinase biosynthesis-activating pheromone (GBAP), triggers the production of the pathogenicity-related extracellular proteases, gelatinase and serine protease, in Enterococcus faecalis. In this study, we synthesized GBAP and its analogs and examined their gelatinase biosynthesis-inducing activity. This study on the structure-activity relationship shows that a lactone ring was indispensable for the activity.     

Structure-activity relationship of gelatinase biosynthesis-activating pheromone of Enterococcus faecalis

The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe⁷ or Trp¹⁰ almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp¹⁰ substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an π-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp¹⁰ was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe⁷ was extended from the core structure into the solvent and was probably directly involved in receptor binding.     

Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism

FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% α-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90°C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated k(d) value of 2μM indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a k(d) value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used.     

A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis

Extracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE^–SprE^–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE⁺SprE⁺). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro- or anti-lytic outcome.     

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.     

Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry.

Cutaneous aging and chronic exposure to UV irradiation leads to alterations in the appearance and biochemical composition of the skin. Members of the MMP family have been involved in the destruction of the extracellular matrix. Among them, gelatinases A and B were found to display elastolytic activity, in vitro. In this study, we first determined the ex vivo elastolytic potential of both endopeptidases, using human skin tissue sections and computerized morphometric analyses, and compared it with those of neutrophil elastase. In such conditions, gelatinase B (50 nM) induced 50% elastolysis. The percentage of elastic fibers degraded by gelatinase A (10-100 nM) never exceeded 10%. Elastolysis by gelatinase B and leukocyte elastase was characterized by a decrease in fiber length and an increase in the average diameter of the fibers. In addition, gelatinase B exhibited fibrillin-degrading activities. On the contrary, gelatinase A (50 nM) elicited up to 50% hydrolysis of collagen fibers, preferentially degrading type III collagen fibers. Gelatinase B did not promote any collagen degrading activity. Our data suggested that in vivo gelatinases could disrupt most extracellular matrix structures of human skin. Gelatinase B and to a much lesser extent, gelatinase A would degrade components of the elastic fibers network while gelatinase A, but not gelatinase B, would alter mostly collagen fibers and also degrade constituents of the dermo-epidermal junction.     

Expression of gelatinases A and B in the ovary of the medaka fish Oryzias latipes.

We cloned cDNAs for gelatinase A and gelatinase B from an ovary cDNA library of the medaka fish Oryzias latipes. The gelatinase A clone encodes a protein of 657 amino acids, whereas the gelatinase B clone encodes a protein of 690 amino acids. Gelatinase A mRNA was expressed in the testis, ovary, intestine, heart, spleen and kidney of the animal. In contrast, gelatinase B mRNA was detected in the ovary. Localization of the respective mRNAs in the ovary was examined using in situ hybridization. Gelatinase A mRNA was found only in the oocytes of small and middle-sized follicles. In contrast, gelatinase B was expressed exclusively in follicular tissues that had ovulated. In situ zymographic analysis revealed that gelatinolytic activity, presumably due to matrix metalloproteinase activity, was detectable in the areas surrounding small and middle-sized follicles, interstitial stromal tissues and the cytoplasm of oocytes. Using extracts of the whole ovary and of ovulated oocytes, several gelatin-degrading enzymes, which probably represent the intermediate and active forms of medaka fish gelatinase A and gelatinase B, were detected by gelatin zymographic analysis. These results clearly indicate that gelatinase A and gelatinase B play a discrete role in the ovary of this lower vertebrate animal.     

The activation of human neutrophil gelatinase

To study the mechanisms of activation of human neutrophil gelatinase, the enzyme has been purified using a combination of chromatography on a DEAE-Sephacel and a gelatin-peptide-Sepharose column. On reducing SDS-polyacrylamide-gel electrophoresis the purified gelatinase ran as a single band of about 94,000 Da, and had a specific activity of 5624.4 units/mg of enzyme protein. When latent gelatinase was treated with trypsin, cathepsin G, neutrophil elastase, HgCl2 or urea, its activity was enhanced and the enzyme was processed and converted into species of the lower molecular mass. Upon activation, the protein band of 94,000 Da of reduced latent gelatinase underwent a decrease of about 6,000-12,000 Da. Formation of the species of lower molecular mass during urea activation could be blocked by the addition of EDTA.     

E. coli proteolytic activity in milk and casein breakdown

Previous studies have focused on both LPS and E. coli experimental mastitis and underlined the respective roles of endogenous proteolysis (including plasmin from the blood stream and other proteases from milk leukocytes), as well as the presence of E. coli in a more intricate system. The aim of this study was to assess the role of E. coli in milk proteolysis and especially that of its proteases in casein breakdown. The first part consisted in the incubation of 104 cfu.mL(-1) of the E. coli strain in raw milk at 37 degrees C for 24 h; the same milk was also incubated with 0.04% sodium azide. Several parameters were evaluated: CFU, plasmin activity, gelatinase activity and pH 4.6 insoluble peptides, including the proportion of gamma-CN. The profile of gelatinase activity was determined by zymography and identified by immunoblotting. In the second part of the study, we examined the profile of CN (alphas-, beta- and kappa-CN) breakdown by E. coli lysate. The results suggest that E. coli proteases have a direct effect on CN, and the increase of gamma-CN in inoculated milk may be generated by both plasmin and the gelatinase. Moreover, the gelatinase activity in the inoculated milk was higher after 24 h of incubation.     

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.     

Quantitative determination of gelatinase activity among enterococci

Gelatinase, hemolysin and aggregation substance have all been reported to be virulence factors of enterococci. In this study, gelatinase production was investigated in isolates of Enterococcus faecalis (n=93), E. faecium (n=49) and E. avium (n=36) recovered from hospitalized patients. Gelatinase was detected in 45% of E. faecalis isolates, but could not be detected in E. faecium and E. avium. Gelatinase activity was then measured by radial diffusion for the 42 gelatinase-positive E. faecalis isolates. To convert gelatinase activity into proteinase K activity, a standard curve was produced by placing different concentrations of proteinase K into wells in the gelatine plate. Gelatinase activity per E. faecalis colony ranged from 2.6 x 10(-7) to 2.2 x 10(-5) microg/ml, proportionate to the activity of proteinase K. An approximately 84-fold difference in gelatinase concentration was observed between the colony producing the highest amount and that producing the lowest amount. This method may be useful for determining the virulence of given isolates in relation to gelatinase production as it is quick, easy and inexpensive to perform.     

The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells

To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor beta1 (TGFbeta1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFbeta1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFbeta1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFbeta1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFbeta1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFbeta1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.     

Identification of a region of beta-amyloid precursor protein essential for its gelatinase A inhibitory activity

Because beta-amyloid precursor protein (APP) has the abilities both to interact with extracellular matrix and to inhibit gelatinase A activity, this molecule is assumed to play a regulatory role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we prepared various derivatives of APP. Functional analyses of proteolytic fragments of soluble APP (sAPP) and glutathione S-transferase fusion proteins, which contain various COOH-terminal parts of sAPP, showed that a site containing residues 579-601 of APP(770) is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP sequence corresponding to residues 586-595 of APP(770) had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion of the NH(2)- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr(588), Asp(591), and Leu(593) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile(586), Met(594), and Pro(595) modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC(50) = 30 nm), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC(50) = 2 microm). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC(50) > 10 microm). The APP-derived inhibitor formed a complex with active gelatinase A but not with progelatinase A, and the complex formation was prevented completely by a hydroxamate-based synthetic inhibitor. Therefore, the decapeptide region of APP is likely an active site-directed inhibitor that has high selectivity toward gelatinase A.     

Cytokines Regulate Gelatinase A and B (Matrix Metalloproteinase 2 and 9) Activity in Cultured Rat Astrocytes

Abstract: Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium-dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1-day-old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24-h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin-1α or -β, or tumor necrosis factor-α for 24 h, a dose-dependent increase in the activity of the 58- and 66-kDa gelatinases and the induction of a 94-kDa gelatinase occurred. All three astrocyte-derived proteases showed maximal activity in the presence of millimolar levels of Ca²⁺, their activity was inhibited in the presence of 1,10-phenanthroline, and their proenzymes were cleaved and activated after incubation with p-aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58-kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94-kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94-kDa gelatinase by lipopolysaccharide was not influenced when interleukin-1 receptor antagonist was included during the 24-h incubation period; however, the antagonist completely blocked interleukin-1β-induced 94-kDa activity and diminished the activity of the 58- and 66-kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin-1β stimulation of the 94-kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte-derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.