Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii

Microbial Pathogenesis 2001; 30: 39–48

Article available online at http://www.idealibrary.com on

doi:10.1006/mpat.2000.0403

MICROBIAL PATHOGENESIS

Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii Byoung-Kuk Naa†, Jae-Chan Kimb & Chul-Yong Songa∗ a

Department of Biology, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea, and bDepartment of Ophthalmology, College of Medicine, Chung-Ang University Medical Center, Seoul 140-757, Korea

(Received August 1, 1999; accepted in revised form September 5, 2000)

A secreted proteinase was purified from the culture supernatant of Acanthamoeba castellanii with several chromatographic steps. The purified proteinase was a chymotrypsin-like serine proteinase. Its molecular weight was approximately 12 kDa on SDS-PAGE, and its native molecular weight was 12 kDa when determined by molecular sieve chromatography. It showed a broad temperature optimum ranging 30–55°C with an optimal at 55°C and an optimal pH of 8.5. It could degrade various protein substrates, such as collagen, fibronectin, laminin, secretory immunoglobulin A, immunoglobulin G, plasminogen, fibrinogen, haemoglobin and rabbit corneal proteins. It showed strong cytopathic effects in cultured cells, including HEp2 and HEK cells. The corneal lesions, induced by both the purified proteinase and A. castellanii, displayed similar clinical results for both cases, in which the stromal infiltration and opacity with the epithelial defect were revealed. These results suggest that the enzyme was highly associated with the pathogenesis of Acanthamoeba. The fact that cytopathic effects and development of corneal lesions caused by the proteinase of Acanthamoeba were inhibited by the proteinase inhibitor suggest that the proteinase inhibitor might be useful as a therapeutic agent.  2001 Academic Press Key words: A. castellanii, proteinase, purification, characterization, keratitis, pathogenesis.

Introduction Acanthamoeba keratitis is a serious vision-threatening inflammatory disease caused by ubiquitous, pathogenic, free-living Acanthamoeba ∗ Author for correspondence: Department of Biology, College of Natural Science, Chung-Ang University, 221 Heuksukdong, Dongjakgu, Seoul 156-756, Korea. E-mail: [email protected] † Present address: Division of Respiratory Viruses, National Institute of Health, Seoul 122-701, Korea. 0882–4010/01/010039+10 $35.00/0

species. Acanthamoeba can directly infect the cornea after trauma associated with contaminated water or contact lens wear, often resulting in serious keratitis [1]. This infection is resistant to therapy, leading to marked visual impairment or even loss of vision [2]. Pathologic studies during an early phase showed destruction of the anterior cornea with infiltration of acute inflammatory cells into the superficial and middle layers of the corneal stroma [3]. At a later phase of Acanthamoeba keratitis, considerable loss of corneal substance had occurred and was followed by ulceration, descemetoceles formation, intense poly 2001 Academic Press

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Purification of proteinase The proteinase of A. castellanii was purified using ammonium sulfate precipitation and a series of chromatographic steps. DEAE Sepharose Fast Flow ion exchange chromatography of the ammonium sulfate precipitates yielded two peaks of proteolytic activity [Fig. 1(a)]. The unbound fractions which showed stronger proteolytic activity were pooled, concentrated and applied to Ultrogel HA adsorption chromatography [Fig. 1(b)]. The active fractions were pooled, concentrated and applied on Sephacryl S-100 HR molecular sieve chromatography [Fig. 1(c)]. Results of the purification step are summarized in Table 1. The molecular weight of the purified enzyme was approximately 12 kDa, as determined by SDS-PAGE (Fig. 2), giving the same estimated size as by Sephacryl S-100 HR molecular sieve chromatography (data not shown). This result indicates that the purified enzyme is monomeric in structure with a single polypeptide chain.

80

0.15

60

0.1

40

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20

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0

0 10 20 30 40 50 60 70 80

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Results

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morphonuclear inflammatory response and finally perforation of the cornea [4, 5]. Until recently, it has been assumed that inflammatory cells, principally neutrophils, were responsible for the pathology of the cornea. However, some evidence suggest the role of secreted proteinases as important virulence factors in Acanthamoeba keratitis [6–8]. For example, the secretory products from trophozoites of Acanthamoeba have collagenolytic activity which induce the damage of collagen shield in an in vitro assay and the rat cornea in vivo [6, 7]. These results implied that Acanthamoeba proteinase is closely related to the pathogenesis of Acanthamoeba by means of destruction of host-protective barriers and promotion of tissue invasion and destruction by this organism. However, thorough investigations about the properties of Acanthamoeba proteinase and its role as a virulence factor have been hampered by the lack of purified proteinase. To establish that the Acanthamoeba keratitis is closely related to the Acanthamoeba proteinase, more detailed studies using purified proteinases are necessary. In this study, we purified a secreted proteinase of A. castellanii, characterized its biochemical properties, and investigated its role as a virulence factor in vitro and in vivo. We also investigated the therapeutic effect of the proteinase inhibitor for the treatment of Acanthamoeba keratitis.

B.-K. Na et al.

0 60

Fraction number

Figure 1. Elution profiles of proteinase of A. castellanii. (a) DEAE Sepharose Fast Flow ion exchange chromatography. (b) Ultrogel HA adsorption chromatography. (c) Sephacryl S-100 HR molecular sieve chromatography. Proteolytic activity (Μ) and absorbence at 280 nm (Β). For detailed explanation, see Materials and Methods.

Optimal pH and optimal temperature Although the purified enzyme was active over a broad pH range, 6.0–10.0, it was most active at 8.5 and showed low activity below 6.0. The

Proteinase of A. castellanii

41

Table 1. Purification scheme of proteinase from A. castellanii Total protein (mg) CFa ASPb DEAEc HAd MSCe

Total activity Specific activity (Unit)f (Unit/mg)f

121.7 87.3 8.6 2.5 0.8

1207.1 1024.7 631.2 478.3 392.1

9.1 11.7 73.4 191.3 490.1

Purification fold

Yield (%)

1 1.3 8.1 21.0 53.9

100 92.6 57.0 43.2 35.4

a

Culture filtrate of A. castellanii. Ammonium sulfate precipitation. c DEAE Sepharose Fast Flow ion exchange chromatography. d Ultrogel HA adsorption chromatography. e Sephacryl S-100 HR molecular sieve chromatography. f One unit of enzyme activity was defined as the amount of enzyme needed to increase 0.1 O.D. unit using azocaseine as the substrate. b

kDa

1

2

200.0 97.4 66.2 45.0

31.0

21.5 14.4

enzyme with inhibitors (Table 2). Serine proteinase inhibitors, DFP and PMSF, significantly inhibited the enzyme activity. An inhibitor of chymotrypsin, TPCK, also reduced the activity. However, no significant inhibition was observed when inhibitors such as TLCK (inhibitor of trypsin-like proteinase), E-64 and iodoacetic acid (inhibitors of cysteine proteinase), pepstatin A (inhibitor of aspartic proteinase) and 1,10-phenanthroline and EDTA (inhibitors of metalloproteinases) were present. While the divalent cations Ca2+ and Mg2+ showed no significant inhibition, Cu2+, Hg2+ and Zn2+ exhibited some inhibitory effects, especially Hg2+. At a concentration of 2 mM of Hg2+, enzyme activity was completely inhibited (data not shown).

6.5

Figure 2. SDS-PAGE analysis of the purified enzyme. Lane 1, standard marker proteins; Lane 2, purified enzyme.

enzyme exhibited a broad temperature optimum, 30–55°C, with maximum activity at 50°C (data not shown). The enzyme was unstable at 50°C and residual activity after 12 h incubation was 22%. At 60°C, the enzyme was completely inactivated after 8 h (data not shown).

Effects of proteinase inhibitors and metal ions The effects of various inhibitors on the activity of enzyme were determined by measuring the residual activity following preincubation of the

Degradations of various proteins The purified enzyme could degrade various protein substrates including fibronectin, collagen, laminin, fibrinogen, plasminogen, IgG, sIgA, BSA, haemoglobin and rabbit corneal proteins (Fig. 3).

Substrate specificity and kinetic study The specificity of the purified proteinase for the synthetic substrate was examined with a series of chromogenic substrates (Table 3). No hydrolysis was detected for L-Leu p-NA, L-Phe p-NA and L-Arg p-NA, indicating that the enzyme had no aminopeptidase-like activity. However, the proteinase showed a high reactivity to N-Suc-AlaAla-Pro-Phe p-NA. When Phe was replaced with

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Table 2. Effects of inhibitors on the activity of purified enzyme Inhibitor

Concentration (mM) Residual activity (%)b

Controla DFP PMSF TPCK TLCK Leupeptin Iodoacetic acid E-64 Pepstatin A 1,10-Phenanthroline EDTA

0.01 0.1 0.01 0.01 0.01 0.01 0.001 0.0001 1 1

100.0 23.1 25.2 73.3 99.3 102.9 105.8 101.2 98.6 93.5 100.8

The effects of inhibitors on the activity of purified enzyme were investigated at three different concentrations of each inhibitor. DFP, TPCK, TLCK, leupeptin and iodoacetic acid (0.01, 0.1 and 0.5 mM); PMSF (0.1, 1 and 5 mM); E-64 (0.001, 0.01 and 0.05 mM); pepstatin A (0.0001, 0.001 and 0.005 mM); 1,10-phenanthroline and EDTA (1, 10 and 50 mM). The concentration giving the highest residual activity is only shown. The other two concentrations also showed similar inhibition pattern without significant difference. a Control represents the activity tested without any inhibitor. b Percentage control.

Met (a P1 site) in the same peptide, proteolytic activity reduced to 26%. High reactivity also resulted when the P1 site was Arg. However, the activity was substantially reduced when the P2 site was Gly (N-Suc-Gly-Gly-Phe p-NA). The apparent Km value of the enzyme was 9.3×10−4 M when determined with N-Suc-Ala-Ala-Pro-Phe p-NA as the substrate (data not shown).

Cytopathogenicity to the cultured cells To confirm that the purified proteinase of A. castellanii is involved in acanthamoebal pathogenicity, we performed an in vitro cytopathogenicity test by using cultured cell systems. Incubation of HEp2 cells with the purified proteinase of A. castellanii resulted in the loss of cell viability within 12 h (data not shown). The morphological change of cells in culture after the addition of purified proteinase is shown in Fig. 4. To eliminate the possibility that these cytopathogenicities are due to other contaminated and minor components, tests using various proteinase inhibitors were carried out simultaneously. The cytopathogenic events were completely inhibited when PMSF-pretreated proteinase was added to HEp2 cells. However, other proteinase inhibitors, such as E-64, pepstatin A and EDTA, did not inhibit the cytopathogenicity. Furthermore, to exclude the pos-

sibility that this cytopathogenicity was due to the toxicity of inhibitors, we performed the cytopathogenicity test using the proteinase inhibitor alone. At the concentrations of various proteinase inhibitors used in the pretreatment of the purified enzyme, each inhibitor alone did not show any significant cytopathogenicity. Thus, these data suggest that the purified proteinase of A. castellanii is closely involved in the cytopathic aspect of HEp2 cells. A similar result was also observed in HEK cells (data not shown).

In vivo pathogenicity test Intrastromal injection of purified proteinase into the cornea of rabbits produced corneal lesions that clinically mimicked those found in rabbit eyes infected with Acanthamoeba by injecting trophozoites of A. castellanii. The corneal opacity with central small epithelial defect or corneal melting were shown in both groups 1 day postinjection. However, inactivated proteinase by pretreatment of PMSF, given simultaneously with the infecting innoculum, did not show any significant corneal lesions (Fig. 5).

Discussion In this study, we purified a secreted proteinase of A. castellanii for its biochemical and pathological

Proteinase of A. castellanii M

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Figure 3. Degradation of various proteins by the purified enzyme. (a) Collagen type I, (b) fibronectin, (c) laminin, (d) immunoglobulin G, (e) secretory immunoglobulin A, (f) plasminogen, (g) fibrinogen, (h) bovine serum albumin, (i) haemoglobin, (j) rabbit corneal extract. Each protein was mixed with purified enzyme (1
g) in 50 mM Tris–HCl buffer (pH 8.5) and incubated at 37°C for 1 and 2 h, respectively. Lane M, standard marker proteins; lane 1, control without enzyme; lane 2, incubated with enzyme for 1 h; lane 3, incubated with enzyme for 2 h.

Table 3. Substrate specificity of the purified enzyme on various chromogenic substrates Substrate L-Leu p-NA L-Phe p-NA L-Arg p-NA Gly-Phe p-NA Gly-Pro-Leu -NA PGLU Phe-Leu p-NA N-Suc-Gly-Gly-Phe p-NA N-Suc-Ala-Ala-Val p-NA N-CBZ-Leu-Leu-Glu -NA N-Acethyl-Ala-Ala-Pro-Ala -NA N-Suc-Ala-Ala-Pro-Phe p-NA N-Suc-Ala-Ala-Pro-Met p-NA N-CBZ-Gly-Gly-Leu p-NA N-Benzoyl-Arg-Gly-Phe-Phe-Leu -NA N-Acethyl-Ile-Glu-Ala-Arg p-NA

Relative activity (%) <0.01 <0.01 <0.01 0.13 0.33 0.22 0.95 0.24 0.12 0.46 100.0 26.43 8.07 2.36 97.56

Dehydrated chromogenic -Naphthylamide (-NA) substrates and p-Nitroanilide (p-NA) substrates (100
M) were incubated with purified enzyme (1
g in 50 mM Tris–HCl, pH 8.5) from A. castellanii at 50°C for 30 min.

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Figure 4. Cytopathic effect of the purified proteinase of A. castellanii on HEp2 cells. Cells were cultured on 24 well plates in the presence or absence of the purified enzyme, with or without the proteinase inhibitor at 37°C for 12 h. (a) Control cells, no proteinase. (b) and (c) HEp2 cells were exposed to the purified enzyme at 1 and 2
g, respectively, resulting in the destruction of monolayer cells. (d) HEp2 cells were exposed to the purified enzyme (2
g) which were treated with PMSF (1 mM). (e) HEp2 cells were exposed to PMSF (10 mM). No cytopathic effect can be seen. Magnification ×100.

Figure 5. Keratitis induced by intrastromal injection of the purified enzyme in pigmented rabbit cornea. (a) and (b) Rabbit cornea injected with the purified enzyme with or without the proteinase inhibitor, PMSF, respectively. (c) and (d) Fluorescein stained rabbit cornea injected with the purified enzyme with or without the proteinase inhibitor, PMSF, respectively.

characterization. The purified proteinase of A. castellanii was a 12 kDa serine proteinase with a single polypeptide chain. Inhibitor and substrate specificity tests indicated that this enzyme belongs to the chymotrypsin-like proteinase family. However, conflicting substrate specificities for Phe and Arg residues at the P1 site should be clarified by solving the three-dimensional structure of the proteinase–substrate complex. It could degrade various proteins such as collagen, fibronectin, laminin, secretory immunoglobulin A (sIgA), immunoglobulin G (IgG), plasminogen,

fibrinogen, fibrin, BSA, haemoglobin and rabbit corneal proteins. Type I collagen is usually considered to be the major collagen of the corneal stroma in most species and play an important role in maintaining corneal integrity. Fibronectin and laminin are glycoproteins present in the human basement membrane as well as in the rat corneal epithelium [9–11]. These proteins promote the attachment of basal epithelial cells to two major integral components of the basal lamina, collagen type IV and heparin sulfate [12]. Proteolysis of these extracellular matrix proteins leads to an end

Proteinase of A. castellanii

stage process of corneal disintegration, since no reparative mechanism exists to restore chain length and function. If degradation exceeds synthesis of these macromolecules, epithelial integrity cannot be maintained, which ultimately leads to necrosis. Therefore, degradation of these structural proteins by the enzyme suggest its close relation to corneal destruction observed in typical Acanthamoeba keratitis by breaking down corneal constituents which normally function as structural proteins. The purified enzyme could degrade immunoglobulins. Immunoglobulins function as a host’s major humoral immunity by preventing or modulating microbial adhesion, invasion and infectivity. sIgA is the major immunoglobulin isotype present in normal tears where it constitutes a front line of defense against microorganisms [13, 14]. It is thought to act primarily as an immunologic barrier, preventing adherence and adsorption of microbes [15]. Inhibition of microorganism’s adherence to the ocular surface would be advantageous because this might diminish the host inflammatory cell response to eliminate microorganisms from the cornea, in turn leading to less innocent bystander destruction of the cornea tissue. It also may prevent colonization by neutralizing microbial pathogens within epithelial cells. The protective role of sIgA in Acanthamoeba infection has been previously reported [16, 17]. sIgA prevent Acanthamoeba infection by inhibiting the binding of the trophozoites to the corneal epithelium. Cleavage of sIgA by the enzyme suggest that it would presumably enhance the ability of the organism to survive on corneal surfaces. Furthermore, the proteinase could degrade IgG, another immunoglobulin isotype contained in tear. Immunization of Acanthamoeba antigen induces the production of parasites specific IgG and delayed-type hypersensitivity responses, but fails to protect the host against subsequent corneal infection with Acanthamoeba [18]. Although the reason of this recrudescence is not yet clear, it may be partially explained by immune escape mechanism through degradation of IgG by Acanthamoeba proteinase. However, more detailed investigations should be performed to clarify the role of Acanthamoeba proteinase in immune escape mechanism of the organism. Acanthamoeba trophozoites produce cytopathic effects on a variety of cultured mammalian cells including Vero, MDBK, HEK, HeLa, PMI and human corneal epithelium [19–21]. Cytopathic effect formed by amoebae appear to be

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due to the liberation of a cytotoxic substance and to actual phagocytosis of the cells by the amoebae [22]. In Acanthamoeba, this cytotoxic substance has been identified as a phospholipase [23]. The purified proteinase of Acanthamoeba exhibited strong cytopathic effects on HEp2 cells and HEK cells. However, the addition of the proteinase inhibitor could completely inhibit its cytopathic effect on cultured cells. This suggests that the cytopathic effect induced by the infection of Acanthamoeba may be closely related with the proteinase of the organism. Intrastromal injection of purified proteinase into the cornea of pigmented rabbits produced corneal lesions that clinically mimicked those found in rabbit cornea with Acanthamoeba keratitis. The corneal lesions caused by this enzyme displayed the stromal infiltration, disintegration and corneal opacity which were very similar to the lesions induced by Acanthamoeba infection. The fact that corneal lesions caused by the enzyme of Acanthamoeba were inhibited by the proteinase inhibitor suggested that it was a main factor which was involved in the pathogenesis of Acanthamoeba and the proteinase inhibitor might be a candidate as a therapeutic agent in the treatment of Acanthamoeba keratitis. In conclusion, Acanthamoeba proteinase was highly associated with pathogenesis of Acanthamoeba by means of destruction of host-protective barriers and promotion of tissue invasion and destruction by this organism. Therefore, the inactivation of the secreted proteinase, either by immunoprohylaxis or proteinase inhibitor, might be useful for treatment of Acanthamoeba infection.

Materials and Methods Organism and culture condition Acanthamoeba castellanii isolated from a patient with Acanthamoeba keratitis was grown axenically at 30°C in peptone-yeast extract-glucose (PYG) medium as previously described [24]. One litre of the culture was centrifuged at 6000×g for 15 min and filtered through a membrane filter (0.22
m, pore size) at 4°C. Ammonium sulfate was added slowly to the supernatant, stirring to a final concentration of 80%. After standing overnight at 4°C, the precipitates were collected by centrifugation at 28 000×g for 15 min, dissolved in 10 ml of phosphate buffered saline (PBS, pH 7.4), and dialysed overnight against distilled water.

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Purification of proteinase The concentrated supernatant was applied to a column (1.6×15 cm) of DEAE Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) which was equilibrated with 50 mM Tris–HCl buffer (pH 8.0) and eluted with the same buffer at a flow rate of 40 ml/h. Adsorbed proteins were eluted with 0.1, 0.2 and 0.3 M NaCl in a stepwise gradient. Eluted fractions were collected (2.5 ml/ fraction) for measurement of absorbence at 280 nm and for enzyme assays. Fractions with proteolytic enzyme activity were pooled, dialysed against distilled water at 4°C and lyophilized. The partially purified enzyme was further purified by using Ultrogel HA adsorption chromatography (Pharmacia; 1.6×15 cm) which was equilibrated with 10 mM sodium phosphate buffer (pH 6.5). Adsorbed proteins were eluted with 10–500 mM sodium phosphate buffer (pH 6.5) in a linear gradient. Proteolytic fractions were pooled, dialysed against distilled water at 4°C and lyophilized. The proteinase was finally purified by using Sephacryl S-100 HR molecular sieve chromatography (Pharmacia; 1.6×80 cm) which was equilibrated with 20 mM Tris–HCl buffer (pH 7.4) containing 0.1 M NaCl. The protein concentration was measured by the method of Lowry et al. [25] using bovine serum albumin (BSA) as the standard.

Enzyme assay Enzyme activity was determined spectrophotometrically following the digestion of azocasein as the substrate. To the enzyme solution, 300
l of 2% azocasein in 50 mM Tris–HCl buffer (pH 8.5) were added and incubated at 37°C for 2 h. The reaction was then stopped by adding 700
l of ice-cold trichloroacetic acid (TCA). The precipitated protein was removed by centrifugation at 10 000 rpm for 3 min and 700
l of the resulting supernatant was mixed with 600
l of 1 N NaOH. The proteolytic activity was determined by measuring the absorbence of the above mixture at 440 nm. One unit of enzyme activity wss defined as the amount of enzyme needed to increase OD440 to 0.1 under above condition.

B.-K. Na et al.

the method of Laemmli [26] using a 15% (w/v) polyacrylamide gel. Standard marker proteins included myosin (200 000), -galactosidase (116 250), phosphorylase B (97 400), serum albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), trypsin inhibitor (21 500), lysozyme (14 400) and aprotinin (6500) (Bio-Rad, Richmond, CA, U.S.A.). Zymography was performed according to the method of Heussen & Dowdle [27].

Determination of native molecular weight The native molecular weight of the purified enzyme was determined by molecular sieve chromatography. The purified enzyme was applied to a column (1.6×80 cm) of Sephacryl S100 HR precalibrated with calibration standards. Standards were aldolase (158 000), bovine serum albumin (67 000), ovalbumin (43 000), chymotrypsinogen A (25 000), ribonuclease A (13 700), and vitamin B12 (1357) (Pharmacia).

Optimal pH and optimal temperature The optimal pH of the purified enzyme was determined in various buffers (ranging pH 5.0–10.0). Thirty
l of the enzyme solution was added to 270
l of each of following buffers: 50 mM sodium acetate buffer (pH 5.0); 50 mM sodium phosphate buffers (pH 6.0–7.0); 50 mM Tris–HCl buffers (pH 7.5–8.5); and 50 mM Glycine–NaOH buffers (pH 9.0–10.0). Then each enzyme solution was incubated for 2 h at 50°C after mixing with 300
l of 2% azocasein as the substrate. For each pH step, blanks were measured separately. To determine the optimal temperature of enzyme activity, 30
l of enzyme solution was added to 270
l of 50 mM Tris–HCl buffer (pH 8.5) and incubated for 2 h at various temperatures ranging 10–70°C with azocasein. For the thermal stability assay, the purified enzyme in 50 mM Tris–HCl buffer (pH 8.5) was preincubated in a sealed tube for various time intervals at 37, 50 and 60°C prior to the proteolytic enzyme assay.

Effects of proteinase inhibitors on enzyme activity SDS-PAGE and zymography Sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) was performed by

The purified enzyme was preincubated at 37°C for 30 min in 50 mM Tris–HCl buffer (pH 8.5) containing each inhibitor. Following the addition of

Proteinase of A. castellanii

substrate, the reaction mixtures were incubated at 50°C for 2 h and each absorbance was measured. The inhibitors used in this study were diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), trans-epoxy-succinyl-L-leucyl-amido-(4-guanidino) butane (E-64), N--tosylL-lysine-chloromethyl ketone (TLCK), N-tosylL-phenylalanine-chloromethyl ketone (TPCK), iodoacetic acid, pepstatin A and ethylenediamine-tetraacetic acid (EDTA). All inhibitors were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). For the effects of metal ions on enzyme activity, the purified enzyme was incubated in 50 mM Tris–HCl buffer (pH 8.5) containing MgCl2, CaCl2, ZnCl2, FeCl4, CuSO4 and AgNO3 (each 2, 5 and 10 mM) at 50°C for 2 h, respectively. Appropriate blanks were treated identically without adding divalent cations.

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N-Succinyl-Gly-Gly-Phe p-Nitroanilide, NAcethyl-Ile-Glu-Ala-Arg p-Nitroanilide, NCBZ-Gly-Gly-Leu p-Nitroanilide, N-SuccinylAla-Ala-Pro-Phe p-Nitroanilide, N-SuccinylAla-Ala-Pro-Met p-Nitroanilide, N-SuccinylAla-Ala-Val p-Nitroanilide and Gly-Pro-Leu Naphthylamide (Sigma). Mixtures of purified enzyme (1
g) and each peptide substrate (final concentration of 100
M) were incubated in 50 mM Tris–HCl buffer (pH 8.5) at 50°C for 30 min. Then, absorbence at 400 nm for p-Nitroanilide and at 340 nm for -Naphthylamide, were measured with a spectrophotometer (Beckman, DU-600). Kinetics were measured with N-Succinyl-Ala-Ala-Pro-Phe p-Nitroanilide as a substrate at concentrations between 5×10−7 and 10−2 M. The enzyme concentration was constant at 10−9 M. The Km was calculated from a Lineweaver–Burk plot of the results.

Cytopathogenic effect of the enzyme Degradation of various proteins Purified collagen (Type I, from bovine achilles tendon), fibronectin (from human plasma), laminin (from human plasma), haemoglobin (from human blood), plasminogen (from human plasma), fibrinogen (from human plasma) and BSA were purchased from Sigma. Immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) were purchased from Cappel Products (Organon Teknica, NV, U.S.A.). These proteins were dissolved in 50 mM Tris– HCl buffer (pH 8.5) at a concentration of 1 mg/ ml. These substrates were incubated with purified enzyme (1
g) for 1 and 2 h at 50°C. The reactions were stopped by adding an equal volume of denaturing sample buffer followed by boiling the mixture for 3 min. SDS-PAGE was performed by the method described above.

HEp2 cells (ATCC CCL23) and HEK cells (Human Embryonic Kidney cells) were cultured in a tissue culture flask (Corning Glass Works, Corning, NY, U.S.A.) with Eagle’s minimum essential medium with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY, U.S.A.). After trypsinization, suspended cells in the same medium were transferred to Nunclone 24 well Multidishes (Nunc A/S, Roskilde, Denmark) at 4×104 cells/well and incubated in a humidified 5% CO2 atmosphere at 37°C. After formation of monolayers, the medium was replaced with serum free medium containing purified enzyme (1 or 2
g/ml) for cultivation. In addition, the effect of the proteinase inhibitor was also tested for the cytopathogenicity assay. The cytopathogenicity assay was carried out by measuring cell growth and observing the morphological changes of cultured cells using a phasecontrast inverted microscope (Olympus, Japan) after 12 h of incubation.

Substrate specificity and kinetic study Substrate specificity of the purified enzyme was determined by using various chromogenic peptide substrates. The peptide substrates for this assay included L-Leu p-Nitroanilide, L-Phe pNitroanilide, L-Arg p-Nitroanilide, Gly-Phe pNitroanilide, pGLU Phe-Leu p-Nitroanilide, NAcetyl-Ala-Ala-Pro-Ala -Naphthylamide, NBenzoyl-Arg-Gly-Phe-Phe-Leu -Naphthylamide, N-CBZ-Leu-Leu-Glu -Naphthylamide,

Intrastromal injection of purified enzyme The pathogenic properties of the purified enzyme were investigated by the intrastromal injection of the purified enzyme into the rabbit cornea. Animals were handled in accordance with the ARVO Statement fo the Use of Animals in Ophthalmic and Vision Research and adhered to the tenets of the Declaration of Helsinki. After

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subconjunctival injection of 0.2 ml of dexamethasone (5 mg/ml) for 4 days into 24 pigmented rabbits, the corneal center was marked using 3 mm length trephine. Purified enzyme (1
g) was injected intrastromally into the right eye of seven rabbits using 31 gauge needles mounted on the microsyringe (1 ml). To evaluate the effect of the proteinase inhibitor, the mixture of the purified enzyme and PMSF was coinjected into the right eye of seven other rabbits using the same method. As experimental groups to evaluate Acanthamoeba keratitis, trophozoites of A. castellanii (2.5×105 cells), with or without PMSF, were injected into four rabbit corneas, respectively. As a control group, normal saline and PMSF were separately injected into two rabbits, respectively. The corneal lesions were observed daily using a slit lamp.

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Acknowledgements 17

This work is supported by a grant of Good Health RND Project (HMP-97-M-5-0055), Ministry of Health and Welfare, Korea.

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