Expression, purification and refolding of recombinant

To produce the recombinant proteins, a fragment harboring a portion of the prodomain and the entire mature domain of nfcpb and nfcpb-L were amplified using the following primers; nfcpb TCCTCTCTTCTCCACGCTAAAT-3’ and

5’-TTAAAGATTTGGAATAGTGTCAG-3’ or nfcpb-L

5’-TTCGATTCTGACTTTTTGAACG-3’ and 5’-TTAAAGTAAAACTCCTGCACCC-3’. The amplified product was purified from gel and subcloned into pEX5-TOPO TA vector (Invitrogen). The nucleotide sequence of cloned cDNA fragments was

determined using an ABI Perkin Elmer 373A automated DNA sequencer (Applied Biosystems) which was then transformed into Escherichia coli BL21 (DE3) cells (Invitrogen). E. coli with pEXP5-NT-TOPO containing NfCPB and NfCPB-L were 1:100 in Luria–Bertani medium with 100 μg/ml of ampicillin and grown at 37°C until the absorbance reached 0.4 ~ 0.6 at 600 nm. Expression was initiated with 1 mM isopropyl β-Dthiogalactopyranoside (IPTG). After, the cells were grown for 4 h at 37°C, pellets resuspended in 8 M urea lysis buffer. The recombinant proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen, Venlo, Netherlands), and monitored by SDS-PAGE.

Refolding of the purified recombinant proteins were performed according to the methods of Kang et al. (2010). Briefly, rNfCPB or rNfCPB-L (each 10 mg) purified by Ni-NTA affinity were slowly added to 500 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM ethylene-diamine-tetra-acetic acid (EDTA), 250 mM L-arginine, 5 mM reduced glutathione (GST) and 1 mM oxidized glutathione (GSSG), and gently stirred at 4℃ for 20 h. To allow processing to an active enzyme, the refolded proteins were further processed as the previous paper (Na et al., 2004). The proteins were dialysed with 10 mM Tris-HCl (pH 7.5) and concentrated with Amicon® Ultra (cut-off 10 kDa; Millipore, Bedford, MA, USA).

D. Production of anti-rNfCPB and anti-rNfCPB-L antibodies

For the production of polyclonal antibody against rNfCPB or rNfCPB-L, the purified and refolded rNfCPB or rNfCPB-L were mixed with an equal volume of complete Freund’s adjuvant (Sigma, St. Louis, MO, USA) and injected intraperitoneally (50 μg/mouse) into 8-weeks-old female BALB/c mouse. The mouse was immunized biweekly for another 4 wk with the each antigen mixed with an equal volume of incomplete Freund’s adjuvant (Sigma). Two weeks after the final booster, the rNfCPB or rNfCPB-L (25 μg /mouse) was injected intravenously without any adjuvant. The mice were sacrificed 2 wk later, and the sera from bloods were collected by centrifugation at 2,500 x g for 30 min at 4℃

and IgG were purified by Protein G affinity column (Incospham, Daejeon, Korea).

Antigen-binding of IgG was analysed by immunoblotting.

E. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting

N. fowleri lysate and ESPs, and NfCPB and NfCPB-L induced by isopropyl-1-thio-β-D-galactopyranoside (IPTG) were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using reducing sample buffer (62.2 mM Tris, pH 6.8; 10% glycerol; 10% 2-mercaptoethanol; 3% SDS;

0.1% bromophenol blue), and stained with coomassie blue. For western blotting, proteins were transferred onto polyvinylidene fluoride (Millipore) membrane for 30 min at 15 V on the Trans-blot^® semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). After, the membranes were blocked with 5% skim milk for overnight at 4°C and reacted with an anti-His antibody (1:4,000 dilutions with 5% skim milk) (Cell Signaling Technology, Inc., Denvers, MA, USA), anti-rNfCPB and rNfCPB-L (1:1,000 dilution with 5% skim milk) were added. After washing PBS containing 0.05% Tween-20 (PBST) three times for 15 min, the membranes were reacted with secondary antibody of a goat anti-mouse IgG conjugated with horse radish peroxidase (1:10,000 dilutions with 5% skim milk, Cell Signaling Technology) for 2 h at 4°C. After washing with PBST three times, the membranes were soaked in enhanced chemical luminescence solution (Amersham Biosciences, Buckinghamshire, England) and exposed to Agfa X-ray film (Agfa-Gevaert N.V., Mortsel, Belgium).

F. Semi-quantitative reverse transcription PCR

To observe the transcriptional patterns of NfCPB and NfCPB-L mRNA in differentiating each stage of N. fowleri, trophozoite and cyst, semi-quantitative reverse transcription (RT) was carried out. To produce the cysticform trophozoites of N. fowleri were incubated with encystment solution (pH 6.8) for 24 h at 37℃.

And then, total RNA were isolated from each of stage of N. fowleri using RNAiso (Takara). Each cDNA was synthesized using a superscript first strand synthesis system according to the manufacture’s recommendation (Invitrogen). Using specific primers for transcription target genes (described above) with equal amounts (30 ng) of each cDNA. RT-PCR was performed to amplify nfcpb and nfcpb-L genes.

G. Enzyme activity assay

For the enzyme activity assay, the hydrolysis of benzyloxycarbonyl-L-phenylalaninyl-L-arginine 4-methyl-coumaryl-7-amide (Z-FR-MCA; Peptide Institute, Osaka, Japan), leucyl-L-arginine-MCA (Z-LR-MCA) and Z-L-arginyl-L-arginine-MCA (Z-RR-MCA), by rNfCPB and rNfCPB-L was fluorometrically observed according to the methods of Na et al. (2008, 2010).

Briefly, 0.2 μg of enzyme solution was added to 190 μl of sodium acetate buffer (pH 4.0 and 7.0) containing 5 μM Z-FR-MCA, Z-LR-MCA and Z-RR-MCA mixture and 10 mM dithiothreitol (DTT), and the release of fluorescence (excitation 355 nm, emission 460 nm) over 30 min at room temperature was assessed with a Fluoroskan Ascent FL (Thermo, Vantaa, Finland). For each pH, the appropriate blank was used as the control group.

H. Biochemical properties of rNfCPB and rNfCPB-L protein

To get fully activated enzymes in the processing of rNfCPB and rNfCPB-L, the pH of refolded samples was firstly adjusted to 4.0 or 5.0 in 3.5 M sodium acetate buffers (pH 2.5) with or without 10 mM DTT. After samples were incubated at 37℃, aliquots were taken every hour, and enzyme activity of rNfCPB and rNfCPB-L were monitored as described above. Then the optimal pH for maximum activity of rNfCPB and rNfCPB-L were observed in sodium acetate (pH 4.0~5.5), sodium phosphate (pH 6.0~6.5) and Tris-HCl (pH 7.0~8.0) buffer. Each 50 nM of rNfCPB or rNfCPB-L was added to each pH buffer containing 5 μM Z-FR-MCA and 10 mM DTT, as which the appropriate blank was separately measured as a control group, and the enzyme activity was measured as described above. To observe the pH stability of rNfCPB or rNFCPB, rNfCPB or rNfCPB were also incubated between pH 4.0 and 8.0 in the appropriate buffers at 37℃.

I. Determination of cysteine protease activity in rNfCPB and rNfCPB-L protein

To assess the cysteine protease activity of rNfCPB and rNfCPB-L, seven kinds of inhibitors, transepoxy-succinyl-L-leucylamido-(4-guanidino)butane (E64), 3-lndoleacetic acid (IAA),

(2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-65

methylbutane ethyl ester (E64D), phenylmethanesulfonyl fluoride (PMSF), EDTA, N-ethylmaleimide (NEM) and pepstatin A (PEP), were treated in the above conditioned media. The proteolytic activity of rNfCPB and rNfCPB-L was observed as described above. And then, active site titration was carried out with a specific inhibitor (E64). The kinetic parameters of rNfCPB or rNfCPB for Z-FR-MCA, Z-LR-MCA and Z-RR-MCA were determined at room temperature, using a fixed amount of enzyme (25 nM) and varying concentrations of substrate in 100 mM sodium acetate (pH 4.5) containing 10 mM DTT.

J. Degradation of host proteins by rNfCPB and rNfCPB-L protein

To assess the degradation of host proteins by rNfCPB and rNfCPB-L, each protein (2 mg/ml) was incubated with refolded rNfCPB or rNfCPB-L (100 nM) in 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.

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)

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

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

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

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

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)

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.

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.

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)

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)

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.

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).

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.

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).

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.

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.

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.

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 - - NH^a - - NH^a

aNH, no hydrolysis

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.

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.

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.

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).

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.

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

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

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.,

문서에서 Characterization of cathepsin B cysteine protease from Naegleria fowleri excretory-secretory proteins exerting immunomodulatory effects (페이지 75-0)