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Oxidation of the mesangial matrix metalloproteinase-2 impairs gelatinolytic activity
J Mattana1, L Margiloff, P Sharma
1Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, NY, USA.
Abstract:
Glomerulosclerosis is characterized by an accumulation of mesangial extracellular matrix. Oxygen radicals are strongly implicated in glomerular injury but it is unclear by what mechanism they could modulate matrix turnover dynamics. We evaluated whether oxidation of the 72 kD mesangial matrix metalloproteinase-2 (MMP-2), the major mesangial matrix-degrading enzyme, could alter its gelatinolytic activity. Oxidation of the MMP-2 using a FeCl3/ascorbate system resulted in impaired ability to degrade [3H]gelatin compared to control. Samples were also subjected to SDS-PAGE gelatin substrate zymography. At the 72 kD position a significant impairment of gelatinolytic activity of oxidized samples was observed, a decrease attenuated by coincubation of samples with the FeCl3/ascorbate system plus the radical spin trap N-tert-butyl-alpha-phenylnitrone suggesting specificity of oxidative changes in the decrease in enzymatic activity. These data represent the first report demonstrating that oxidation of the MMP-2 diminishes its activity and suggest a previously undescribed mechanism by which oxygen radicals may contribute to altered turnover of extracellular matrix.
Insights
Oxidative stress impairs the activity of matrix metalloproteinase-2 (MMP-2), a key enzyme in glomerular extracellular matrix turnover. This finding reveals a novel mechanism linking oxygen radicals to glomerulosclerosis progression.
Area of Science:
- Nephrology
- Biochemistry
- Cell Biology
Background:
- Glomerulosclerosis involves mesangial extracellular matrix accumulation.
- Oxygen radicals are implicated in glomerular injury.
- The mechanism by which radicals affect matrix turnover is unclear.
Purpose of the Study:
- To investigate if oxidation of mesangial matrix metalloproteinase-2 (MMP-2) affects its gelatinolytic activity.
- To explore a potential mechanism for oxygen radical-induced glomerular injury.
Main Methods:
- Oxidation of purified MMP-2 using a FeCl3/ascorbate system.
- Assessing MMP-2 gelatinolytic activity using [3H]gelatin degradation assays.
- Analyzing MMP-2 activity via SDS-PAGE gelatin substrate zymography.
- Utilizing radical spin trap N-tert-butyl-alpha-phenylnitrone to confirm specificity.
Main Results:
- Oxidized MMP-2 showed significantly impaired ability to degrade gelatin compared to controls.
- SDS-PAGE gelatin zymography confirmed reduced gelatinolytic activity of oxidized MMP-2.
- Radical spin trap partially attenuated the decrease in activity, suggesting oxidative modification specificity.
Conclusions:
- Oxidation diminishes the enzymatic activity of MMP-2.
- This study presents a novel mechanism where oxygen radicals contribute to altered extracellular matrix turnover in glomerulosclerosis by inhibiting MMP-2.