A novel and simple immunocapture assay for determination of gelatinase-b (MMP-9) activities in biological fluids: Saliva from patients with Sjögren's Syndrome contain increased latent and active gelatinase-b levels

doi:10.1016/S0945-053X(98)90116-0

Matrix Biology

Volume 17, Issues 8–9, December 1998, Pages 657-665

https://doi.org/10.1016/S0945-053X(98)90116-0 Get rights and content

Abstract

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys↓IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLueGly↓IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can measured directly using a chromogenic peptide substrate for urokinase. The assay has been specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this body MMp-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed.

We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: $17.0 ± 4.9 vs 12.2 ± 2.5 × 10^{4} cpm/ml$ ( $p > 0.05$ , and 44.0 ( $4.0 vs 36.1 ± 1.9 × 10^{4} cmp/ml$ ( $p > 0.05$ ), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients $8.9 ± 2.5 U/mg$ , controls $1.0 ± 0.5 U/mg$ ( $p = 0.002$ ); latent plus active MMP-9: patients $53.1 ± 9.8 U/mg$ , controls $16.5 ± 2.6 U/mg$ ( $p = 0.01$ ).

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Allosteric Communications between Domains Modulate the Activity of Matrix Metalloprotease-1
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An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intradomain dynamics occurring at picosecond to millisecond timescales. However, the correlation between interdomain dynamics and the function of a protein is poorly understood. Here, we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type 1 collagen fibrils correlate with its activity. Using single-molecule Förster resonance energy transfer, we identified functionally relevant open conformations in which the two MMP1 domains are well separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. We fitted the histograms of single-molecule Förster resonance energy transfer values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We used a two-state Poisson process to describe the dynamics and calculate the kinetic rates from the fit parameters. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an interdomain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single-molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single-molecule tracking can overcome for other insoluble substrates. Interdomain communications are likely important for multidomain proteins.
Activation of MMP-9 activity by acrolein in saliva from patients with primary Sjögren's syndrome and its mechanism
2017, International Journal of Biochemistry and Cell Biology
We have recently reported that the altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially responsible for autoimmune diseases in patients with primary Sjögren’s syndrome (pSS). In the current study, it was found that the specific activity (activity/ng protein) of metalloproteinase-9 (MMP-9) in saliva was elevated about 2.4-fold in pSS patients. Accordingly, it was examined whether MMP-9 is activated by acrolein. It was found that the MMP-9 with 92 kDa molecular weight was activated by acrolein. Under the conditions studied, Cys99, located in the propeptide, was conjugated with acrolein together with Cys230, 244, 302, 314, 329, 347, 361, 373, 388 and 516, which are located in fibronectin repeats and glycosyl domains, but not on the active site of MMP-9. In addition, 82 and 68 kDa constructs of MMP-9s, lacking the NH₂-terminal domain that contains Cys99, were not activated by acrolein. The results suggest that acrolein conjugation at Cys99 caused the active site of MMP-9 to be exposed. Activation of MMP-9 by acrolein was inhibited by cysteine, and slightly by lysine, because these amino acids inhibited acrolein conjugation with MMP-9. Conversely, MMP-9 activity in the presence of 50 μM acrolein was enhanced by 100 μM histidine. This was due to the inhibition of acrolein conjugation with His405 and 411 located at the Zn²⁺ binding site of MMP-9. These results suggest that activation of 92 kDa MMP-9 by acrolein is involved in tissue damage in pSS patients and is regulated by cysteine and histidine, and slightly by lysine. Activated 82 and 68 kDa MMP-9 s were not detected in saliva of pSS patients by Western blotting.
Increase in acrolein-conjugated immunoglobulins in saliva from patients with primary Sjögren's syndrome
2015, Clinica Chimica Acta
We previously reported that the level of protein-conjugated acrolein (PC-Acro), a marker of cell or tissue damage, was increased in saliva from patients with primary Sjögren's syndrome (pSS), and that the level of PC-Acro was well correlated with the severity of pSS.
Acrolein-conjugated immunoglobulins were measured in saliva from pSS patients.
The activities of autoantibodies recognizing Sjögren's syndrome SSA (Ro) and SSB (La) proteins in saliva from pSS patients were approximately 3- to 5-fold higher than those from control subjects. We also found that autoantibody activities recognizing SSA (Ro) and SSB (La) proteins increased after acrolein treatment of saliva from control subjects. When an antibody against human serum albumin was treated with acrolein, the ability to recognize albumin was reduced but the ability to recognize other proteins was increased. Twenty-four and eleven kinds of acrolein-conjugated amino acids were found at the variable and constant regions of peptides, respectively, obtained from the immunoglobulins in saliva from pSS patients.
The altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially involved in autoimmune diseases.
Plasma active matrix metalloproteinase 9 and indices of diastolic function in patients with preserved systolic function
2013, International Journal of Cardiology
Citation Excerpt :
Furthermore, substrate zymographic techniques for assessing activity are known to interfere with TIMP binding [7], therefore ‘active’ MMP levels determined by zymography likely overestimate the true in vivo activity of these enzymes. This study utilised an alternative assay system, which measures the ability of antibody captured MMPs to cleave a fluorescently tagged substrate, resulting in the quantification of endogenous MMP activity [8]. Previously, we found that in a retrospective cohort of patients with stable coronary artery disease, there was an association of elevated active plasma MMP 9 level with increased degree of diastolic dysfunction by conventional echocardiography [9].
This study aimed to investigate whether the endogenous active levels of MMP-9 or tissue inhibitor of metalloproteinases-1 (TIMP-1) were related to indices of diastolic dysfunction (DD) in the setting of contemporary treatment of coronary artery disease (CAD).
We prospectively studied 116 patients with CAD and preserved left ventricular LV systolic function (ejection fraction ≥ 45%). All patients were free of heart failure symptoms at recruitment and underwent percutaneous intervention (PCI) of culprit lesions. Demographic and angiographic characteristics were collected. Plasma samples were analysed for the active form of MMP-9 and TIMP-1 using enzyme-linked immunosorbent assay-based isoform sensitive assays. Conventional and tissue Doppler-echocardiographic assessment of diastolic filling was undertaken with measurements of maximal early (E) and late (A) transmitral velocities in diastole, E/A ratio, E-wave deceleration time, isovolumic relaxation time, peak systolic (S), diastolic (D) and atrial reversal velocities of pulmonary venous flow, S/D fraction, time difference between A and duration of atrial reversal flow, early diastolic peak velocities of the lateral mitral annulus (E′) and E/E′. Active MMP-9 level was higher in patients with more severe phases of DD (normal [n = 22]: median 0.57 ng/ml; mild [n = 19] 0.83 ng/ml; mild-moderate [n = 41] 0.64 ng/ml; moderate or severe [n = 34] 1.63 ng/ml; p < 0.0001 for trend). Three month post-PCI elevated levels of active MMP-9 had an adjusted odds ratio of 11.2 (2.3–56.0, p < 0.004) for association with moderate or severe DD.
Elevated active MMP-9 level is associated with more severe DD in patients with CAD and preserved systolic function, which may indicate abnormal extracellular matrix metabolism in myocardial ischaemia.
Targeting TNF-α suppresses the production of MMP-9 in human salivary gland cells
2013, Archives of Oral Biology
Tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine that plays an essential role in inflammation and apoptosis. Our previous study suggested that TNF-α-induced activation of matrix metalloproteinase-9 (MMP-9) resulted in the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS) via disruption of the acinar cell-basement membrane. Recently, a wide array of biological agents has been designed to inhibit TNF, including etanercept and adalimumab.
In this study, we demonstrate the suppressive effect of anti-TNF agents on TNF-α-induced MMP-9 production in NS-AV-AC, an immortalized human salivary gland acinar cell line.
NS-AV-AC cells were treated with etanercept or adalimumab after TNF-α treatment. MMP-9 production and enzymatic activity were, respectively, visualized by real-time PCR and ELISA assay, and evaluated by gelatin zymography, and apoptosis was evaluated by DNA fragmentation assay.
TNF-α induced the production of MMP-9 in NS-SV-AC cells. However, this production was greatly inhibited by treatment with etanercept or adalimumab. In addition, TNF-α-induced DNA fragmentation was prevented by treatment with etanercept or adalimumab.
These results may indicate that anti-TNF agents would have therapeutic efficacy for preventing destruction of the acinar structure in the salivary glands of patients with SS.
Protease profiling of different biofluids in type 1 diabetes mellitus
2012, Clinical Biochemistry
Citation Excerpt :
In an attempt to disclose the proteolytic events underlying type 1 diabetes’ pathogenesis and its related complications, a combined approach with zymography-nano‐LC-MS/MS was applied to the bodily fluids serum, saliva and urine. Besides the most used fluids for clinical purposes, saliva was included in the study attending to its increasingly recognized potential for early diagnosis of diseases [26,27], as already tested in oral and systemic pathologies, with some works reporting increased proteolytic activity in periodontitis, Sjogren's syndrome and acute myocardial infarction [28–30]. For the best of our knowledge this is the first study where a deep characterization of proteases underlying type 1 diabetes and related complications like retinopathy and nephropathy was performed simultaneously in serum, urine and saliva.
We aimed to disclose the proteolytic events underlying type 1 diabetes and related complication through protease profiling in the bodily fluids serum, urine and saliva.
Zymography followed by LC-MS/MS was performed for protease identification and quantitative comparison of proteolytic activity between healthy, type 1 diabetic patients with no complications and with retinopathy and nephropathy. Western blotting was also accomplished for MMP-9 and MMP-2 identification and expression analysis.
Only MMP-2 and MMP-9 were observed in serum with significantly increased levels and activity observed in diabetic patients. In urine and saliva other proteases besides MMPs were identified by MS and presented disease-dependent activity variations. Among these are complex MMP-9/Neutrophil gelatinase-associated lipocalin, aminopeptidase N, azurocidin and kallikrein 1 with more activity noticed in type 1 diabetes patients with nephropathy and/or retinopathy.
Our data highlight the usefulness of urine and saliva for the monitoring of type-1 diabetes-related proteolytic events, where aminopeptidase N, azurocidin and kallikrein 1 appear as promising screening targets for type 1 diabetes‐related complications.

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Regular paperA novel and simple immunocapture assay for determination of gelatinase-b (MMP-9) activities in biological fluids: Saliva from patients with Sjögren's Syndrome contain increased latent and active gelatinase-b levels

Abstract

Fibrinolysis

Pharmacology & Therapeutics

Cell

J. Hepatol.

J. Biol. Chem.

Clinica Chimica Acta

Serum mettalloproteinases and their inhibitors: markers for malignant potential

British Journal of Cancer

Convenient fluorometric assay for matrix mettalloprotienase activity and its application in biological media

FEBS Letters

Matrix-metalloproteinases: a review

Crit. Rev. Oral Biol. Med.

Regular paper
A novel and simple immunocapture assay for determination of gelatinase-b (MMP-9) activities in biological fluids: Saliva from patients with Sjögren's Syndrome contain increased latent and active gelatinase-b levels