Acanthamoeba castellanii Castellani (ATCC #30011; Manassas, Virgina, USA) was cultured axenically in peptone-yeast-glucose (PYG) medium at 25°C. Encystation was induced as previously described [21]. Briefly, approximately 5×10⁵ cells in post-logarithmic growth phase were collected, washed with phosphate-buffered saline (PBS), and incubated in 10 ml of encystation medium (95 mM NaCl, 5 mM KCl, 8 mM MgSO₄, 0.4 mM CaCl₂, 1 mM NaHCO₃, and 20 mM Tris-HCl, pH 9.0) for 72 hr. The morphological change from trophozoites into cysts was observed by microscopy. Encystation efficiency was calculated by counting cysts after treating cells with 0.05% sarkosyl and 0.22% trypan blue, which selectively stains nonviable cells [21,22].

A. castellanii mRNA was isolated from trophozoites using an Oligotex mRNA purification kit (Qiagen, Valencia, California, USA). Single-stranded cDNA was synthesized using the BD SMART™ RACE cDNA amplification kit (BD Biosciences, Palo Alto, California, USA) followed by the manufacturer’s instructions. A partial sequence of AcCP gene was amplified by PCR from cDNA using degenerate oligonucleotide primers designed based on conserved amino acids flanking the active sites of the presently known cathepsin L-family cysteine proteases. The forward primer was 5′-TGYGGWTCGTGYTGGTCATT-3′ and the reverse primer was 5′-CCAAGTGTTCYTGACYRTCCA-3′. The amplification reaction was done with a thermal cycling profile of 94°C for 4 min, 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, followed by a 72°C extension for 10 min. The PCR product was analyzed on 1.2% agarose gel, gel-purified and ligated into the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA). The ligation was transformed into Escherichia coli DH5α competent cells and positive clones were screened by colony PCR for the presence of plasmid with the appropriate insert. A total of 20 clones were randomly selected and the nucleotide sequences of the inserts were determined. As the result, partial gene sequence encoding for AcCP, which did not match any presently known genes encoding Acanthamoeba cysteine proteases, was obtained. To obtain a full-length sequence of AcCP, rapid amplification of cDNA ends (RACE) procedures were performed using the BD SMART™ RACE cDNA amplification kit (BD Biosciences). Following synthesis of the first strand cDNA, the second strand cDNA was synthesized and ligated to the cDNA adaptor. The 5′- and 3′-RACE were carried out with gene-specific primers (GSPs) and an adaptor primer (AP1). GSPs were designed based on the AcCP partial sequence obtained from the PCR with degenerate primers. GSP1 for the 5′-RACE was 5′-CTCAGAGAGCGAGACCAGAGTGCCACG-3′ and GSP2 for 3′-RACE was 5′-ACTGAGAACGGCCAGGATTACTGGCTC-3′. The PCR products were gel purified, ligated, transformed and sequenced. Primary structure analysis of the deduced amino acid sequences and phylogenetic analysis were conducted with DNASTAR (DNASTAR, Madison, Wisconsin, USA), PSORT (http://www.psort.org/) and Signal P (http://www.cbs.dtu.dk/services/SignalP/). The phylogenetic tree was constructed using the neighbour-joining method with MEGA4 (http://www.megasoftware.net). Bootstrap proportions were used to assess the robustness of the tree with 1,000 bootstrap replications. The gene encoding AcCP was deposited in GenBank with an accession number of EF053509.

To produce rAcCP protein, a fragment harboring a portion of the prodomain and the entire mature domain of AcCP was amplified using the following primers: 5′-GGATCCGAGGAGTTTGTCTTCAGGTGG-3′, which harbors a 5′ BamHI site and 5′-AAGCTTTTAAGCGGTGGGGTAGGAGGC-3′, which contains a 5′ HindIII site. The purified PCR product was ligated into the pGEM-T Easy vector (Promega) and was transformed into E. coli DH5α. The resulting plasmid DNA was digested with the appropriate restriction enzymes, ligated into the pQE-30 expression vector (Qiagen), which was then transformed into E. coli M15 (pREP4) cells (Qiagen). The selected clones were cultured in Luria-Bertani broth supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin at 37°C and rAcCP expression was induced with isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM for 3 hr at 37°C. The cells were harvested by centrifugation, suspended in 8 M urea lysis buffer and incubated overnight at room temperature. The supernatant was collected by centrifugation and the rAcCP was purified from the supernatant with nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) according to the manufacturer’s instructions. Purification and purity of the recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Refolding of the purified recombinant protein was performed as describe previously [23,24]. Ni-NTA affinity purified rAcCP (1 mg) was slowly added to 100 ml of 100 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, 250 mM L-arginine, 5 mM reduced glutathione (GST), 1 mM oxidised glutathione (GSSG) and gently stirred at 4°C overnight. The sample was dialysed against 10 mM Tris-HCl (pH 7.5) and concentrated with Centricon Plus (cut-off 10 kDa; Millipore, Billerica, MA, USA). The resulting rAcCP was further processed in different pH buffers (pH 4.0 to 7.0) to analyze the optimal pH condition required for processing to fully matured enzyme as describe previously [24]. In brief, the refolded rAcCP was incubated in different pH buffers (pH 4.0 to 7.0). The aliquots were taken at the indicated times and were analyzed by SDS-PAGE and enzyme activity assay. The enzyme activity of rAcCP was assayed fluorometrically as the hydrolysis of benzyloxycarbonyl-L-leucyl-L-arginine-MCA (ZLR-MCA; Peptide Institute, Osaka, Japan). Fluorescence was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, using a Fluoroskan Ascent FL (Thermo, Vantaa, Finland).

Mouse polyclonal antibody specific for AcCP (anti-AcCP) was produced by immunizing mice with purified rAcCP protein. Briefly, purified rAcCP protein (100 μg) was intraperitoneally injected into 2 8-week-old BALB/c mice (Bionics, Seoul, Korea) with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, Missouri, USA). Before immunization, blood samples were collected from the mice for control experiments. Two boosters with rAcCP (100 μg) mixed with an equal volume of Freund’s incomplete adjuvant (Sigma) were administered at 14 and 28 days after the first injection, respectively. Blood was obtained from the heart of mice at 7 days after administration of the final booster, and anti-AcCP were obtained from the blood via centrifugation for 15 min at 4°C and analyzed its reactivity against rAcCP by Western blotting.

The optimal pH for rAcCP activity was determined in sodium acetate (pH 3.5–5.5), sodium phosphate (pH 6.0–6.5), and Tris-HCl (pH 7.0–7.5). For these experiments, 50 nM rAcCP was added to each buffer containing 5 μM Z-LR-MCA and 10 mM DTT. Appropriate pH blanks were included as controls. To examine the pH stability of rAcCP, the enzyme was incubated at 37°C in the appropriate buffer (pH 4.0–8.0), and residual enzyme activity was measured as described above. The kinetic parameters of rAcCP for Z-LR-MCA, Z-L-phenylalaninyl-L-arginine-MCA (Z-FR-MCA) and Z-L-arginyl-L-arginine-MCA (Z-RR-MCA) were determined at room temperature, using a constant amount of enzyme (30 nM) and varying concentrations of substrate in 100 mM sodium acetate (pH 4.0) containing 10 mM DTT. The concentration of rAcCP was determined by active site titration with trans-epoxy-succinyl-L-leucylamido( 4-guanidino)-butane (E-64). The kinetic constants Km and Vmax were determined using GraphPad software (GraphPad Software Inc., La Jolla, California, USA).

To assess the degradation of human proteins by rAcCP, various proteins (2 mg/ml) were separately incubated with rAcCP (100 nM) in 50 mM sodium acetate (pH 4.0) or Tris-HCl (pH 7.0) in the presence of 1 mM GSH for 3 hr at 37°C. The proteins used for hydrolysis assay were hemoglobin (from human blood), albumin (from human serum), immunoglobulin G (IgG; from human serum), IgA (from human serum) and fibronectin (from human plasma), all purchased from Sigma. After the reactions were terminated by adding reducing sample buffer, protein degradation was analyzed by SDS-PAGE.

To determine the cellular localization of AcCP, the AcCP gene was cloned into the pGAPDH-EGFP vector, which contains an Acanthamoeba glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter for constitutive expression and an enhanced green fluorescent protein (EGFP) reporter gene [25]. The AcCP gene was amplified by PCR with primers that incorporated an NdeI restriction site at the 5′-end (5′-GGCATATGTACAAGCATGACCCGCTTAC-3′) and an SpeI restriction site at the 3′-end (5′-GGACTAGTAGCGGTGGGGTAGGAGGC-3′), and the PCR product was cloned into the pGAPDH-EGFP. The resulted plasmids (pGAPDH-AcCP-EGFP) was then transfected to A. castellanii trophozoites followed by the method described previously [26]. Briefly, approximately 4×10⁵ cells/well were seeded into a 6-well culture plate containing 3 ml of PYG medium and incubated at 25°C overnight. Plasmid DNA (4 μg) in 100 μl of amoeba culture medium was mixed with 20 μl of SuperFect transfection reagent (Qiagen), incubated for 10 min at room temperature, and then added to the cells. After incubation at 25°C for 3 hr to allow for uptake of the DNA-SuperFect complexes, the cells were washed with PBS, incubated for 24 hr, and placed in encystation medium and incubated for 24, 48, or 72 hr [27]. The AcCP-EGFP expression in the amoeba at each time point was assessed by fluorescence microscopy. The cells were also counter-stained with LysoTracker Red DND 99 (Molecular Probes, Eugene, Oregon, USA), which labels acidic compartments such as lysosomes and autophagolysosomes, and the fluorescence was measured by using band-pass filters that provided excitation and emission wavelengths of 500 to 530 nm and 570 to 590 nm, respectively.

To further analyze expression pattern of AcCP in A. castellanii, western blot analysis for different stages of the amoeba was performed. Encystation of trophozoites was induced by the method described above [21,22]. The amoeba cells were collected at 24, 48, or 72 hr after encystation induction. Lysates from A. castellanii trophozoites and cysts collected at each time point were separated by SDS-PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked overnight with 5% skim milk in Tris-buffered saline (TBS) at 4°C, washed with TBS containing 0.05% Tween 20 (TBST), and incubated with anti-AcCP (1:1,000) for 2 hr at room temperature. After washing with TBST several times, the membranes were incubated with anti-mouse horseradish peroxidase-labeled IgG (1:2,000; Dako, Glostrup, Denmark) for 30 min at room temperature. Proteins were detected with enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham Biosciences, Piscataway, New Jersey, USA). Membranes were stripped and probed with a mouse monoclonal anti-actin antibody (1:5,000; Santa Cruz Biotechnology, Santa Cruz, California, USA) for 2 hr to detect Acanthamoeba actin as a loading control [27]. Membranes were washed with TBST and incubated with anti-mouse horseradish peroxidase-labeled IgG (1:2,000; Dako) for 30 min at room temperature and the proteins were detected with ECL reagents (Amersham Biosciences). Western blot analysis for cultured medium collected at 24, 48, or 72 hr incubation of trophozites was also performed with anti-AcCP to analyze secretion of AcCP to culture medium as the same method described above.

The rAcCP, comprising a portion of the prodomain and entire mature domain, was expressed in E. coli as an insoluble protein, with an approximate molecular weight of 36 kDa, which is coincided with estimated molecular mass from the deduced amino acid sequences (Fig. 2A). The purified rAcCP was refolded under alkaline conditions and further incubated in different pHs to allow auto-catalytic processing. The refolded rAcCP was processed to the enzymatically-active species of approximately 30 kDa under acidic conditions, pH 4.0 and 5.0 (Fig. 2B, C). The fully processed rAcCP was effectively inhibited by a cysteine protease inhibitor, E-64 (data not shown). The fully activated rAcCP showed hydrolytic activity in acidic conditions with an optimum at pH 4.0 (Fig. 3A). The enzyme was relatively stable at neutral pH values, but it was unstable under acidic pHs (Fig. 3B). The hydrolytic activity of rAcCP for several human proteins, including hemoglobin, albumin, IgG, IgA and fibronectin, was analyzed. The recombinant enzyme effectively hydrolyzed all tested proteins under acidic conditions (pH 4.0), but did not substantially degrade the proteins under neutral conditions (pH 7.0) (Fig. 3C). Only fibronectin was partially hydrolyzed by rAcCP at neutral pH. The substrate specificity of rAcCP was characterized with several fluorogenic dipeptidyl substrates. The enzyme showed strong activity against Z-FR-MCA and Z-LR-MCA, but did not against Z-RRMCA (Table 1).

Cellular localization of AcCP was determined using an Acanthamoeba vector expressing AcCP with a C-terminal EGFP fusion (AcCP-EGFP). A. castellanii trophozoites were transfected with the plasmid and then examined by fluorescence microscopy followed by counter staining with LysoTracker Red. The AcCP-EGFP fusion protein was distributed in multiple vesicle-like structures in the cytoplasm of both trophozoites and cysts, which were colocalized with the LysoTracker-labeled compartments, indicating that AcCP was localized in lysosomes (Fig. 4). Western blot analysis using anti-AcCP was performed to evaluate the expression profile of AcCP in different developmental stages of amoeba, trophozoites and cysts. The results indicated that AcCP expression was observed in both trophozoites and cysts with a maximum at trophozoite stage (Fig. 5). Western blot analysis for cultured medium revealed that AcCP was detected in cultured medium of trophozoites (Fig. 5). These results collectively suggested that AcCP was expressed in all developmental stages of the Acanthamoeba and was secreted or released from the trophozoites.