Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient

Abstract

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, wepurified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Koreankeratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequentialchromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDaprotease had a pH optimum of 8.5 and a temperature optimum of 55?C. Phenylmethylsulfonylfluoride and 4-(2-Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activityof the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degradedvarious extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine proteasesecreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis,such as in corneal tissue invasion, immune evasion and nutrient uptake.