Article

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: 0.97). 01/2004; 41(4):189-96. DOI: 10.3347/kjp.2003.41.4.189
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
47 Views
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.
    Parasitology Research 10/2008; 104(1):185-90. DOI:10.1007/s00436-008-1185-z · 2.33 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    The Korean Journal of Parasitology 01/2007; 44(4):321-30. DOI:10.3347/kjp.2006.44.4.321 · 0.97 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Acanthamoeba are opportunistic protozoan parasites that can cause painful, visionthreatening keratitis. However the pathogenesis and pathophysiology of Acanthamoeba keratitis remain incompletely understood. Most cases of Acanthamoeba keratitis develop as a result of poor hygiene in contact lens care but it is unclear how amoebae transmigrate from the environment into the cornea leading to inflammation, photophobia and blindness. Acanthamoeba keratitis has become increasingly important in the past few decades due to increasing populations of contact lens users. The mechanisms associated with the pathogenesis of Acanthamoeba are highly complex, depending on the virulence properties of the parasite, host susceptibility and the environmental conditions. Complete understanding of Acanthamoeba pathogenesis and its associated risks factors should allow us to design strategies for disease prevention and for the rational development of therapeutic interventions against these devastating infections. Acanthamoeba keratitis has become a significant problem in recent years, especially in contact lens wearers exposed to contaminated water.
Show more

Preview

Download
0 Downloads
Available from