50 mM sodium acetate buffer (pH 4.5) with 10 mM DTT for 3 h at 37℃. The purified collagen (from human placenta), fibronectin (from human plasma), hemoglobin (from human blood), albumin (from human serum), IgA (from human serum), IgG (from human serum) and IgM (from human serum) (Sigma) were used as target protein. After the reactions were terminated by adding reducing sample buffer, the degradation was analyzed by SDS-PAGE.
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K. MTT Assay
As mouse BV2 cells were cultured as monolayer in DMEM containing 5%
FBS (Gibco), 1% penicillin/streptomycin (Gibco) and 1% L-glutamine (Welgene) at 37°C, 5% CO2 concentration. The details of this experiment using 96 well cell culture plate (Nunc A/S, Roskilde, Denmark) were as follow: 2ⅹ104 cells were treated with 6.25, 12.5, 25, 50, and 100 μg /ml of NfCPB, NfCPB-L or mixture of NfCPB and NfCPB-L at 37℃ for 24 h. The supernatant was discard from plate incubated with 100 μl of MTT solution (1 mg/ml of concentration) (Sigma), for 3 h at 37℃ and then 100 μl of DMSO (Sigma) was added. The reactant was used at 595 nm with ELISA reader.
L. Statistical analysis
All data of fluorogenic enzyme activity represented the mean value of percent activity± standard deviation (mean±SD). The data determined by student’s t test.
(p<0.05, each)
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III. Result
To construction of N. fowleri ESTs data base, I cloned cDNA library from N.
fowleri trophozoites and performed sequencing analysis of 500 randomly selected clones. Each N. fowleri EST was compared with the predicted proteins by BLAST analysis. In addition, the ESTs contain several cathepsin genes (8 genes) in the N.
fowleri trophozoite (Table 1).
Table 1. Classification of the annotated clusters of N. fowleri ESTs
Classification Nunmber of clusters
AMPK-activated protein kinase gamma-2 subunit 1
PfkB family carbohydrate kinase 1
Heat shock protein 20 6
Hsp C2 heat shock protein 2
proly-Trna synthetase 1
Ras GTPase 1
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Vacuolar ATP synthase protein 1
Vacuolar ATP synthase subunit 1
ATPase 2
ATP-ADP translocator 2
ADP-ribosylation factor 1 1
GTP-binding nuclear protein ran 1
NADP-dependent isocitrate dehydrogenase 1
Translation initiation factor Eif-2B alpha subunit 1
Proteasome subunit alpha type-4 1
Guanine nucleotide-binding protein subunit beta 1
26S proteasome regulatory subunit Mts4 1
Trifunctional enzyme alpha subunit 1
Chaperonin containing TCP1 subunit 4) 1
Proteasome subunit alpha 1
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Proteasome alpha3 subunit 1
Ubiquitin precursor 1
Polyubiquitin 2
Ubiquitin specific protease 14 1
Ubiqiutin-like protein 1
Calreticulin 1
Translation elongation factor 2 1
Ras-related GTP-binding protein 1
Elongation factor 1 gamma 1
Regulator of G-protein signaling 13 1
Cytochrome c1 1
Small G-protein 1
Cytochrome B5 1
GTPase-like protein 1
BTB-POZ domain-containing protein 1
Glioma tumor suppressor candidate region gene 1
High mobility group protein putative 1
Alpha-2-macroqlobulin-like protein2 1
RAB1A, member RAS oncogene family 1
Elongation factor 1-delta 1
Calmodulin 1
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DNA RNA binding protein 1
Cytochrome b5 1
COMM domain containing 3 1
Zinc finger(C3HC4-type RING finger) family protein 1
DUF75 family 1
Elongation factor 1-alpha 1
Translation elongation factor 2 1
Shwachman-Bodian-Diamond protein-like protein 1
TM2 domain-containing protein 1
Rho quanidine dissociation inhibitor 1
Bax inhibitor family protein 1
DNA-binding protein-related 1
Surfactant B protein 1
Rho guanidine dissociation inhibitor 1
Translation elongation factor 2 1
No putative protein 49
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A. Cloning and sequence analysis of nfcpb and nfcpb-L gene
After the construction of cDNA library from N. fowleri trophozoites and the sequencing analysis of 500 randomly selected clones performed by RT-PCR with gene specific primers and RACE-PCR, we obtained a complete cDNA sequence encoding a novel cysteine protease of nfcpb and nfcpb-L gene, respectively (Fig. 1).
The open reading frame consisted of 1,038 bp and 939 bp sequence, and encoded 345 and 313 amino acids (molecular weight were 38.4 kDa and 34 kDa), respectively. The nucleotide sequences of nfcpb and nfcpb-L have been deposited in GeneBnak database with accession number KJ159026 and KJ159027. The nfcpb amino acids, Gln-123, was through to be associated with the oxyanion hole, and Cys-129, His-283 and Asn-303 were to be associated with the cysteine protease active site (cysteine, histidine and asparagine residues) (Fig. 1a). The nfcpb-L amino acids, Gln-110, Cys-116, His-257 and Asn-279 were similar with nfcpb, and the occluding loop was characterized by two adjacent histidine residues (Fig. 1b).
Amino acid sequence alignment analysis revealed that the sequence of nfcpb or nfcpb-L and its homologues from other organisms shared an overall conserved construction of a typical cathepsin B or cathepsin B-like (Fig. 2). On the results of phylogenetic analysis with various cathepsin B or cathepsin B-like protease, nfcpb and nfcpb-L made a close cluster with N. gruberi cathepsin B or cathepsin B-like gene, respectively (Fig. 3)
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Fig. 1. Full-length cDNA sequences of nfcpb (a) and nfcpb-L (b). The start codon (ATG) and stop codon (TAA) are in bold face. The glutamine residue of the oxyanion hole was a circle. The active sites of cysteine, histidine, and asparagine residues were box.
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Fig. 2. Multiple amino acid sequence alignment of NfCPB (a) and NfCPB-L (b).
The glutamine residue of the oxyanion hole was an inverted triangle. The active sites of cysteine, histidine, and asparagine residues were black inverted triangle.
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Fig. 3. Phylogenetic analysis of amino acid sequences of nfcpb and nfcpb-L with various organisms. The tree was built by neighbor-joining method using the MEGA 6 program. Distance on the x-axis represents the grade of sequence homology, and distance of the Y-axis are arbitrary.
S.japonicum_(CAX71086)
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B. Differential expression of NfCPB and NfCPB-L in N. fowleri
To observe the differential expression of the nfcpb and nfcpb-L gene, semi-quantitative RT-PCR using RNAs isolated from two differential stages of N.
fowleri was carried out. NfCPB and NfCPB-L expression were detected throughout trophozoite stage, but did not in cyst stage (Fig. 4)
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Fig. 4. Semi-quantitative RT-PCR for detection of nfcpb (b) and nfcpb-L (C) transcription in N fowleri cysts (Cy) and trophozoites (Tr). PCR products were analyzed on 1.2% agarose gel with ethidium bromide staining. A. Quantity of each total RNA samples (a) was equal.
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C. Expression, purification and refolding of rNfCPB and rNfCPB-L protein
After a portion of the prodomain and the entire mature domain of nfcpb or nfcpb-L were amplified, cloned into the pEX5-NT-TOPO expression vector, and transformed into E.coli, rNfCPB and rNfCPB-L as insoluble proteins showed the molecular mass of 38.4 kDa or 34 kDa, respectively (Fig. 5). The recombinant proteins were purified by Ni-NTA affinity chromatography and then refolded under alkaline conditions (Fig. 5a, c). As the results of western blot using anti-NfCPB or anti-anti-NfCPB-L antibody, the Nf-ESPs and lysate were strongly reacted with them (Fig. 5b, d).
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Fig. 5. Expression, purification and refolding of rNfCPB and NfCPB-L protein. a rNfCPB protein was analyzed by SDS-PAGE. Lane 1, IPTG uninduced Escherichia coli lysate; lane 2, IPTG induced E. coli lysate; lane 3, Purified rNfCPB; lane 4, Refolded rNfCPB. b NfCPB expression analysis by western bolt using anti-NfCPB antibody. Lane 1, N. fowleri ESPs; lane 2, N. fowleri lysate. c rNfCPB-L protein was analyzed by SDS-PAGE. Lane 1, IPTG uninduced E. coli lysate; lane 2, IPTG induced E. coli lysate; lane 3, Purified rNfCPB-L lane 4, Refolded rNfCPB-L. d rNfCPB-L expression analysis by western blot using anti-NfCPB-L antibody. Lane 1, Nf-ESPs; lane 2, N. fowleri lysate.
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D. Biochemical characterization of rNfCPB and rNfCPB-L protein
To confirm the proteolytic activity of rNfCPB and rNfCPB-L, the conventional enzyme assay using fluorogenic peptide substrates was carried out (Fig. 6). Firstly, on the results of the pH profiles of the rNfCPB and rNfCPB-L protease activity, the optimal pH were between pH 4.5 and pH 6.5, and the optimal pH for maximum activity of rNfCPB and rNfCPB-L was pH 4.5 (Fig. 6a, b). In addition, the rNfCPB and rNfCPB-L showed the proteolytic activity on Z-FR-MAC and- Z-LR-MCA, but did not hydrolyzed Z-RR-MCA (Fig. 6a, b). The rNfCPB was relatively stable at pH 4.0 to pH 8.0, while enzyme stability was decreased in time-dependent conditions (Fig. 7a). The stability of rNfCPB-L didn’t be changed at pH 4.0 to pH 7.0, but decreased at pH 8.0 (Fig. 7b). On the results of inhibition test for the proteolytic activity of rNfCPB and rNfCPB-L as cysteine proteases, they were completely inhibited by cysteine protease inhibitors, 64, E-64D, and IAA (Fig. 8a, b). In addition, both rNfCPB and rNfCPB-L appeared high catalytic ability on Z-FR-MCA and Z-LR-MCA, but showed little or no activity against Z-RR-MCA (Table 2).
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Fig. 6. The pH profiles of enzyme activity of rNfCPB (a) and rNfCPB-L (b) protein. The enzyme activity of rNfCPB and rNfCPB-L on the three different fluorogenic peptide substrate (Z-FR-MCA, Z-LR-MCA and Z-RR-MCA) were measured in 100 mM sodium acetate (pH 4.0 ~6.5) and Tris-HCl (pH 7.0~8.0) buffer supplemented with 10 mM DTT.
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Fig. 7. Enzyme stability of rNfCPB (a) and rNfCPB-L (b) protein. Each enzyme incubated with 100 mM sodium acetate (pH 4.0 ~6.0) and Tris-HCl (pH 7.0~8.0) buffer supplemented with 10 mM DTT at 37℃ for the indicated time. Residual enzyme activities were performed with Z-LR-MCA as a substrate.
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Fig. 8. Proteolytic activity of rNfCPB (a) and NfCPB-L (b) protein as cysteine protease by incubating with various cysteine protease inhibitors. Following the addition of each inhibitor, the enzyme activity of rNfCPB and rNfCPB-L on the fluorogenic peptide substrate (Z-LR-MCA) were measured in 100 mM sodium acetate (pH 4.5) buffer supplemented with 10 mM DTT.
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Table 2. Kinetic parameters for substrate hydrolysis by rNfCPB and rNfCPB-like protein
Substrate NfCPB NfCPB-like
Km (μM) kcat (s-1) kcat/Km (s-1M-1) Km (μM) kcat (s-1) kcat/Km (s-1M-1)
Z-FR-MCA 65.6 0.318 4.82 × 103 35.8 0.476 13.31 × 103
Z-LR-MCA 60.8 0.327 5.38 × 103 28.7 0.512 17.86 × 103
Z-RR-MCA - - NHa - - NHa
aNH, no hydrolysis
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E. Degradation of host proteins
As the results of proteolytic activity of rNfCPB and rNfCPB against several human IgA, IgG and IgM, all substrates were readily hydrolyzed by rNfCPB or rNfCPB-L at acidic pH (Fig. 9). And also, other human proteins such as collagen (Fig. 10a), fibronectin (Fig. 10b), hemoglobin (Fig. 10c) and albumin (Fig. 10d) were hydrolyzed by rNfCPB or rNfCPB-L at acidic pH.
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Fig. 9. The proteolytic ability of rNfCPB and rNfCPB-L protein on various human immunoglobulins. IgA (a), IgG (b) and IgM (c) were incubated with rNfCPB (lane 2) and rNfCPB-L (lane 3) in 100 mM sodium acetate (pH 4.5) buffer with 10 mM DTT for 3 h at 37℃, and analyzed by SDS-PAGE. Each lane 1, control Ig without any enzymes.
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Fig. 10. Degradation of various human proteins by the proteolytic activity of rNfCPB and rNfCPB-L protein. Collagen (a), fibronectin (b), albumin (c) and hemoglobin (d) were incubated with rNfCPB (lane 2) and rNfCPB-L (lane 3) in 100 mM sodium acetate (pH 4.5) buffer with 10 mM DTT for 3 h at 37℃, and analyzed by SDS-PAGE. Each lane 1, control protein without any enzymes.
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F. rNfCPB and rNfCPB-L induces cell death of mouse BV2 cells
We investigated the connection between cell damage effect and inhibition of microglial proliferation by rNfCPB or rNfCPB-L treatment on BV2 cells. The cells were treated with several concentrations of rNfCPB and rNfCPB, and the viability of cells were assessed by MTT assay. As shown in Figure 2, the treatment of BV2 microglial cells with rNfCPB and rNfCPB-L markedly reduced cell viability in dose dependent manners (none, 6.25, 12.5, 25, 50 and 100 μg/ml) and the highest effect was observed after treating 24 h (Fig. 11).
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Fig. 11. Viability of BV2 cells treated with different concentration of rNfCPB (a), rNfCPB-L (b) and combined NfCPB with NfCPB-L (c) protein. Both NfCPB and NfCPB-L increased cell death in BV2 microglial cells. Cells were treated with different concentrations (none, 6.25, 12.5 25, 50 and 100 μg/ml) of each enzymes and then cell death patterns of BV2 cells were measured by MTT assay. Cell viability calculated by the percent of control.
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IV. Discussion
PAM development due to N. fowleri infection is initiated by the introduction of amoebic trophozoites in contaminated water into the nasal cavity of host. Then, N. fowleri trophozoites attach to the nasal mucosa, migrate along the olfactory nerves, cross the blood-brain-barrier, and enter the forebrain. But, the nutrient uptake, neutralization mechanism and evasion of host immune systems, and penetration of host tissue have not been elucidated clearly (Ma et al., 1990;
Visvesvara et al., 2007).
Various parasitic cysteine proteases are important virulence factors for parasitic infection. In relation with the pathogenicity of N. fowleri, we have previously reported that N. fowleri lysate and ESPs containing various pathogenic proteins such as secreting effector proteins, cysteine protease including cathepsin B and cathepsin B-like protease, secretory lipase, peroxiredoxins, and thrombin receptors, which function in amoebic entering into host cell and as various dominant antigenic proteins, were involved in amoebic pathogenesis (Shin et al., 2001; Kim et al., 2008, Kim et al., 2009).
In this study, we obtained full-length sequence of cathepsin B and cathepsin B-like cysteine protease of N. fowleri and characterized the biochemical and functional properties of these two enzymes. The open reading frame consists of
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1,038 bp and 939 bp (encodes 345 and 313 amino acid), and molecular weight of NfCPB and NfCPB-L were 38.4 kDa and 34 kDa by SDS-PAGE analysis and well reacted with each anti-antibody by western bot analysis, respectively. By multiple alignment analysis, they showed 56% and 46% identity with N. gruberi cathepsin B and cathepsin B-like enzyme, respectively (data not shown). Phylogenetic analysis revealed that NfCPB and NfCPB-L were closely related majority of N.
gruberi CPB or CPB-L protease. Although the CPB enzymes including conserved motifs of cysteine protease showed 43~53% identity to homologues from other translation (Coulombe, 1996). We produced rNfCPB and rNfCPB-L enzymes using the E. coli expression system, and recombinant proteins were purified with Ni-NTA resin and refolded under various conditions, and assessed their biochemical properties.
The substrate specific preference of rNfCPB and rNfCPB-L for Z-FR-MCA and Z-LR-MCA were observed (did not reacted with Z-RR-MCA), as which it showed in C. sinensis CPB (Na et al., 2010). As the confirmation of two enzymes in the optimal pH and pH stability under different pH condition, rNfCPB and
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rNfCPB-L showed its optimum activity at acidic pH 4.5. Full-activated rNfCPB and rNfCPB-L were incubated in various pH buffers for the indicated time, and residual enzyme activity showed enzyme stability on acidic pH but unstable on neutral pH until 3 h.
Other typical biochemical features for cysteine proteases of C. sinensis cathepsin F including sensitivity against E64 and the requirement of a reducing condition for maximum activity were also previously reported (Na et al., 2006; Na et al., 2008; Kang et al., 2010). In this study, rNfCPB and rNfCPB-L are clearly inhibited their activity by cysteine specific inhibitor, E64 activated by dithiothreitol (DTT) and 2-mecaptoethanol, as which showed in other amoebic CPB (Que and Reed, 2000). In our experiments, two enzymes were pre-incubated with two fold diluted DTT as cysteine protease activator, and the enzyme activity did not affected by various DTT concentrations (Data not shown).
The cysteine protease of pathogenic amoeba, E. histolytica, play an important role for supporting to attachment by degrading the intestinal mucosa and penetration of host tissue by digesting extracellular matrix, degrading host protein for evasion from host immune response, activating host cell proteolytic cascades such as complement and assistance to produce metastatic lesions (Keene at al., 1986). Moreover, CPB of Eimeria tenella was related with cell invasion by sporozoites (Rieux et al., 2012), and T. gondii CPB located to rhoptries, secretory organelles required for parasite invasion into cells (Que et al., 2002). L. mexicana
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cathepsin B-like cysteine protease (CPC)-deficient mutant showed that infectivity to macrophages was reduced by CPC null mutant of promastigote form and infected low percentage of the cells in vitro. The CPC gene led to modest defects in lesion development in vivo. It suggested that the primary role of the CPC protease is to facilitate promasigote invasion (Bart et al., 1997).
On the other hand, cathepsin B proteases clearly play an important role in the biology of flukes (Smooker et al., 2010). It has been shown that a cathepsin protease inhibitor can reduce schistosome burdens in the murine model and that RNA interference resulted in the stunting of parasite growth (Correnti et al., 2009).
F. hepatica CPB is very important for the survival and functions of newly excysted juvenile form, including playing a role in invasion through the gut wall (Beckham et al., 2009). In addition, at early stage after F. hepatica infection, its CPB is also released into host tissues and highly antigenic in animals both during infection (Law et al., 2003). F. gigantica cathepsin B2 expressed in early stages may be involved in digestion of host connective tissues and evasion of host immune systems during their penetration and migration (Chantree et al., 2012). In addition, cathepsin B3 in F. gignatica was confirmed as the stage-specific expression (Sethadavit et al., 2009). On the other hand, CPB of Ancylostoma cannium may play roles in nutrient digestion (Harrop et al., 1995), and then, the expression of CPB was more abundant in eggs and larval of A. caninum, as which might play role in the early development of A. caninum (Yang et al., 2011).
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As the present results of semi-quantitative RT-PCR in two differential stages of N. fowleri to observe the differential expression of the nfcpb and nfcpb-L gene, NfCPB and NfCPB-L expression were detected throughout trophozoite stage, but did not in cyst stage. It suggest that amoebic trophozoites, active forms of N.
fowleri, show more pathogenic activity than cystic form. In this present result, the ability of hydrolysis by rNfCPB and rNfCPB-L to various human proteins including collagen, hemoglobin, albumin, fibronectin and immunoglobulins was assessed, and two enzymes could hydrolyze the above proteins. This result suggests that rNfCPB and rNfCPB-L may be associated with the attachment on host tissue (proteolysis of collagen and fibronectin), evasion in host immune systems (proteolysis of immunoglobulins) and nutrient uptake (proteolysis of albumin and hemoglobin), and thus play an important role in the pathogenesis of N. fowleri. In the future study, the more detailed functional activity of NfCPB and NfCPB-L in the pathogenic mechanism of N. fowleri will be carried out.
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V. Conclusion
Naegleria fowleri causes a lethal PAM in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 bp and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 kDa and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56% and 46% identity to N. gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 kDa and 34 kDa bands on SDS-PAGE and western-blot analysis using their respective antibodies.
Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximal at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.
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