Purification and characterization of a secretory glutamic acid-specific endopeptidase from Staphylococcus aureus sp. strain C-72

Abstract

A glutamic acid-specific endopeptidase (Glu-protease) was purified and characterized from the culture supernatant of Staphylococcus aureus (S. aureus) sp. strain C-72. For the purification, ammonium sulfate precipitation (30 - 70% cut-off), HiTrap-Q anion exchange and HiPrep 16/60 Sephacryl S-200 HR gel filtration column chromatographies were employed in order. The purified protease designated to as SA72 appeared as a single polypeptide with apparent molecular weight of 36.4 kDa, as judged by SDS-polyacrylamide gel electrophoresis and gel filtraion column chromatography. The SA72 protease could cleave casein and hydrolyzed the synthetic chromogenic substrate L-2135 to liberated p-nitroanilline (pNA), indicating that it is a Glu-specific endopeptidase. The optimal temperature and the pH range for SA72 protease was 50℃ and pH 6.0-6.5, respectively. The enzyme was stable at the temperature up to 40℃, but protease activity significantly decreased at 60℃. It was revealed by periodic acid-Schiff (PAS) staining that the SA72 protease is not a glycoprotease. The activity of SA72 protease could be inhibited by DFP and PMSF, suggesting that it belongs to the family of serine protease. The N-terminal amino acid sequence of SA72 protease showed approximately 95% homology to those of bacterial Glu-specific proteases. The SA72 protease actively cleaved zymogens such as plasminogen, fibrinogen, and prothrombin. Even FXa was also sensitive to the digestion of SA72 protease. However serum proteins such as g-globulin and bovine serum albumin (BSA) showed a resistance to the SA72 cleavage. These results suggest that the SA72 protease may target the zymogens and digest the activated proteases that are involved in hemostatic process. Interestingly, the SA72 protease digested fibrin polymer with 0.005 plasmin unit per ㎍ protein, as shown by fibrin plate assay. The fibrinolytic activity of SA72 protease was also confirmed by turbidity assay. When the fibrin polymer was incubated with SA72 protease, the turbidity of fibrin polymer solution decreased in a time-dependent manner. These results strongly suggest that the SA72 protease can actually digest the cross-linked fibrin. Taken together the results presented in this study showed also well matched to the fact that S. aureus secrets a potent non-proteolytic activator of the blood coagulation zymogen prothrombin and the prototype of a newly established zymogen activator and adhesion protein (ZAAP) family.