Abstract
We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.
Highlights
The plasminogen/plasmin system plays crucial roles in blood clot lysis and in various physiological and pathological events including inflammation, tissue remodeling, tumor metastasis, and angiogenesis, where localized proteolysis is required (1–5)
1 The abbreviations used are: u-PA, urokinase-type plasminogen activator; Activated partial thromboplastin time (APTT), activated partial thromboplastin time; BCEC, bovine capillary endothelial cell; BL-angiostatin, angiostatin-like plasminogen fragment produced by BL-MA cleavage; BL-MA, bacillolysin MA; Val[79] bonds to yield mature plasmin, which consists of two polypeptide chains that are held together by disulfide bridges
Purification and Primary Structure of BL-MA—B. megaterium A9542 was selected from a screen for searching modulators of plasminogen activation, employing 1,040 strains of microorganisms
Summary
Materials—The following chemicals and proteins were from commercial sources. Val-Leu-Lys-pNA (H-Val-Leu-Lys-p-nitroanilide) was from Bachem (Bubendorf, Switzerland). For determination of proteolytic activity, BL-MA (0.1 nM to 37 M or concentrations shown in each experiment) was incubated with appropriate substrate protein solution (200 g/ml or concentrations given in each experiment) in TBS/T containing 1 mM CaCl2 at 37 °C for 60 min (or time shown in each experiment). Human plasminogen (2 M) was incubated with 16 nM BL-MA at 37 °C for 60 min in 8 ml of TBS/T containing 1 mM CaCl2. After 30 min, HUVECs that had been starved overnight in serum- and growth factor-free EGM and 0.1% bovine serum albumin were seeded into the upper chamber (1.1 ϫ 105 cells in 500 l of EGM containing 20 g/ml BL-angiostatin or angiostatin K1–3) and incubated for 5 h. Plasma clot formation was initiated by adding 60 l of 133 mM CaCl2, and thrombelastogram was recorded at 37 °C
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