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

Department of Neurology, Ulsan University College of Medicine, Ulsan 680-060, Republic of Korea.
The Korean Journal of Parasitology (Impact Factor: 1.15). 01/2004; 41(4):189-96. DOI: 10.3347/kjp.2003.41.4.189
Source: PubMed


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

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    • "To examine the ability of E/S products to degrade hemoglobin, assays were performed as described previously by Kim et al. (2003), with slight modifications. Briefly, 20 μg of E/S products were incubated with 20 μl of 0.1% hemoglobin (Sigma) solution in 0.1 M phosphate buffer (pH 5.5) for 16 h at 37°C. "
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    • "more, by adding 0.1 mM PMSF, the CPE of 3 Acanthamoeba strains with different degree of virulence came to be equal level. This indicates that the difference in virulence and cytopathy have may originated mainly from PMSF susceptible serine proteinases as suggested by previous studies (Leher et al., 1998; Alfrei et al., 2000; Kong et al., 2000; Kim et al., 2003). Many serine proteinases with different molecular size had been reported in Acanthamoeba (Mitra et al., 1995; Cho et al., 2000; Kong et al., 2000; Na et al., 2001; Hurt et al., 2003). "
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    ABSTRACT: The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.
    Full-text · Article · Jan 2007 · The Korean Journal of Parasitology
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    • "For example, Hadas and Mazur (1993), demonstrated the presence of a 35 and a 65 KD serine proteases from eight species of Acanthamoeba. Later studies revealed the presence of 43, 59, 70 and 100-130 KD cysteine proteases and 33, 42, 47, 60, 75, 100 and 133 KD serine proteases in Acanthamoeba (Alfieri et al., 2000; Cho et al., 2000; Kong et al., 2000; Kim et al., 2003; Leher et al., 1998). Other studies have shown the presence of additional 12, 107 and 230 KD serine proteases (Cao et al., 1998; Khan et al., 2000; Na et al., 2001), as well as other hydrolytic enzymes such as elastase (Ferrante and Bates, 1988), phospholipase A (Cursons & Brown, 1978; Mishra et al., 1985; Victoria & Korn, 1975) and there is some indication of metalloprotease activities in Acanthamoeba (Mitro et al., 1994). "
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    Preview · Article · Jul 2004
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