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J. Amaral, L. Notari, W. Stetler-Stevenson, P. Becerra; Gelatinase A/MMP-2-Mediated Proteolysis of Pigment Epithelium-Derived factor (PEDF) is pH Dependent. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2966.
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© ARVO (1962-2015); The Authors (2016-present)
The antiangiogenic and neurotrophic factor PEDF is a member of the SERPIN superfamily of proteins. We have shown before that the majority of proteinases cleave PEDF (50-kDa) at its homologous serpin reactive loop leaving an active limited product of 46-kDa, and that gelatinases (MMP-2 and MMP-9) can degrade PEDF abolishing its biological activities. In this study we characterized the MMP-2-mediated proteolysis of PEDF.
PEDF and gelatinaseA/MMP-2 were purified. Enzymatic assays of MMP-2 activity were against itself or PEDF as substrates. Proteolysis was carried out with 0.5 - 1 µg of substrate at 37°C for 0 - 24 hours with protease:substrate ratios of 1:1, 1:10 or 1:100 (w:w) incubated in phosphate buffer (4 mM Na/K-phosphate pH 7.4, 150 mM NaCl and 10 mM CaCl2) or Tris buffer (50 mM Tris/MES pH 4 - 8, 150 mM NaCl, 5 mM CaCl2, and 1 µM ZnCl2). The reactions were stopped with 10 mM EDTA. The overall conformation of the PEDF protein was examined by controlled proteolysis performed with a chymotrypsin:PEDF ratio of 1:100 (w:w) incubated at 25°C for 0 - 90 min. Products were resolved by SDS-polyacrylamide gel electrophoresis followed by Coommassie blue staining. Gelatinolytic activity was also assayed by zymography.
MMP-2 in the phosphate buffer degraded PEDF completely, but MMP-2 in the Tris pH 7.5 buffer only cleaved PEDF to a 46-kDa product. Controlled chymotryptic proteolysis of PEDF in the above buffers produced a 46-kDa polypeptide as a limited product, revealing no change in the overall conformation for the PEDF protein in these buffers. MMP-2 in the phosphate buffer autoproteolyzed, as seen by SDS-PAGE and gelatin zymography, but MMP-2 in the Tris buffer pH 7.5 did not change its electrophoretic migration. Given that the pH of the phosphate buffer decreased with increasing concentrations of CaCl2, the effect of pH on MMP-2 activity was examined. MMP-2 in Tris/MES buffers had optimum activity at pH 5.5 - 6, with maximum MMP-2 autoproteolysis and PEDF degradation at this pH range.
These results demonstrate MMP-2 autoactivation and PEDF degradation in a pH-dependent fashion, and suggest in vivo regulation of antiangiogenic and neurotrophic PEDF activities mediated by pH changes.
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