title: Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing.
abstract: OBJECTIVE
  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.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Wu
authors.forename: S P
authors.initials: SP
authors.lastname: Becerra
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000502:physiology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015843:Serpins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology; D014822:Vitreous Body / Q000737:chemistry / Q000201:enzymology*; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D007611:Aprotinin; D005136:Eye Proteins; D009414:Nerve Growth Factors; D010436:Pepstatins; D011506:Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D013450:Sulfones; D000099174:Pigment Epithelium-Derived Factor; C014195:Streptomyces pepsin inhibitor; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; C031375:pepstatin