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, 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.

Fig. 1

Chromatograms of proteinase purification from the culture supernatant of Acanthamoeba lugdunensis KA/E2. A. CM-Sepharose chromatography of the ammonium sulfate-precipitated culture supernatant. B. Chromatogram of Sephacryl S-200 column of the enzymatically active fractions (fractions 36-42) from A. C. Chromatogram of enzymatically active fractions (fraction numbers 28-31) of B from a mono Q anion-exchange column.

Fig. 2

SDS-PAGE of the purified enzyme in a gradient acrylamide gel. A single protein band (arrow) stained with Coomassie blue was observed with a size of approximately 33 kDa. Lane: M, standard size marker: P, purified proteinase.

Fig. 3

Effect of pH and temperature on serine proteinase activity. A. The enzyme activity was assayed in sodium acetate (pH 5.5-6.0), phosphate (pH 6.0-8.0), or Tris-HCl (pH 8.0-10.0). B. Optimum temperature of the enzyme. Enzyme activity is expressed as a percentage of the maximum proteolytic activity observed.

Fig. 4

Substrate specificity of the purified secretory serine proteinase of A. lugdunensis KA/E2. M, protein size standard; Lane 1, reaction mixture without enzyme; 2-4, incubation for 30 sec, 10 min, 30 min; M, protein size standard. Marks indicate α-chains of type I collagen (▶) monomers (➡) and dimers (⇨) of hemoglobin.

Fig. 5

Degradation of IgG and IgA by the serine proteinase of A. lugdunensis KA/E2. Lane 1, reaction mixture without enzyme; 2-4, incubation for 30 sec, 10 min, 30 min; M, protein size. The heavy (▶) and light chains (▷) of immunoglobulins are indicated.