Y Q Wu1, S P Becerra. 1. Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
Abstract
PURPOSE: Experiments were designed to identify proteolytic activities that cleave pigment epithelium-derived factor (PEDF), a member of the serpin (serine protease inhibitor) family. METHODS: Proteins in vitreous humor from bovine eyes were analyzed by Western blot with antiserum to human recombinant PEDF protein. Protein fractionation was by ammonium sulfate saturation and by S-Sepharose column chromatography. Proteolytic activities were determined by gelatin zymography and by solution assays against PEDF or chromogenic peptide substrates. RESULTS: PEDF protein was identified and purified to near homogeneity from vitreous humor of bovine eyes. Limited proteolysis showed that the vitreal protein has a protease-sensitive region at its serpin-exposed peptide loop. Proteolytic activities that cleave the PEDF 49.5 kDa-polypeptide were identified only when proteins from these extracts were separated by 45% to 70% ammonium sulfate fractionation (P70). The degradation product had an apparent molecular weight of 46 kDa. This result is consistent with cleavage at the serpin-exposed loop. The PEDF-cleavage activity in P70 was inhibited specifically by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), but not by aprotinin, EDTA, or pepstatin. The vitreal P70 extracts contained 49- and 53-kDa gelatinolytic activities that also were inhibited by AEBSF and not by EDTA, aprotinin, or pepstatin. The PEDF-cleavage activity did not hydrolyze substrates for thrombin, factor Xa, alpha-chymotrypsin, trypsin, or plasmin, nor did it immunoreact with antibody to urokinase plasminogen activator. CONCLUSIONS: These data indicated that vitreous has a serine-proteolytic activity associated with a novel 49/53-kDa enzyme that cleaves the PEDF protein in a serpinase fashion. In addition to cleavage in vitro, these proteases might play a role in modulating PEDF in vivo.
PURPOSE: Experiments were designed to identify proteolytic activities that cleave pigment epithelium-derived factor (PEDF), a member of the serpin (serine protease inhibitor) family. METHODS: Proteins in vitreous humor from bovine eyes were analyzed by Western blot with antiserum to human recombinant PEDF protein. Protein fractionation was by ammonium sulfate saturation and by S-Sepharose column chromatography. Proteolytic activities were determined by gelatin zymography and by solution assays against PEDF or chromogenic peptide substrates. RESULTS:PEDF protein was identified and purified to near homogeneity from vitreous humor of bovine eyes. Limited proteolysis showed that the vitreal protein has a protease-sensitive region at its serpin-exposed peptide loop. Proteolytic activities that cleave the PEDF 49.5 kDa-polypeptide were identified only when proteins from these extracts were separated by 45% to 70% ammonium sulfate fractionation (P70). The degradation product had an apparent molecular weight of 46 kDa. This result is consistent with cleavage at the serpin-exposed loop. The PEDF-cleavage activity in P70 was inhibited specifically by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), but not by aprotinin, EDTA, or pepstatin. The vitreal P70 extracts contained 49- and 53-kDa gelatinolytic activities that also were inhibited by AEBSF and not by EDTA, aprotinin, or pepstatin. The PEDF-cleavage activity did not hydrolyze substrates for thrombin, factor Xa, alpha-chymotrypsin, trypsin, or plasmin, nor did it immunoreact with antibody to urokinase plasminogen activator. CONCLUSIONS: These data indicated that vitreous has a serine-proteolytic activity associated with a novel 49/53-kDa enzyme that cleaves the PEDF protein in a serpinase fashion. In addition to cleavage in vitro, these proteases might play a role in modulating PEDF in vivo.
Authors: S Patricia Becerra; L Alberto Perez-Mediavilla; John E Weldon; Silvia Locatelli-Hoops; Preenie Senanayake; Luigi Notari; Vicente Notario; Joe G Hollyfield Journal: J Biol Chem Date: 2008-09-19 Impact factor: 5.157