Protein Expression and Purification 63 (2009) 26–32

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Protein Expression and Purification

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Purification, refolding and autoactivation of the recombinant cysteine proteinaseEhCP112 from Entamoeba histolytica

Laura I. Quintas-Granados a, Esther Orozco b, Luis G. Brieba c, Rossana Arroyo b, Jaime Ortega-López a,*

a Department of Biotechnology and Bioengineering, Centro de Investigación y Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco,CP 07360 Mexico City, Mexicob Department of Infectomic and Molecular Pathogenesis, Centro de Investigación y Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco,CP 07360 Mexico City, Mexicoc National Laboratory of Genomics for Biodiversity (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN (CINVESTAV-IPN), Campus Guanajuato,AP 629 Irapuato, Guanajuato CP36500, México

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 June 2008and in revised form 7 September 2008Available online 14 September 2008

Keywords:Entamoeba histolyticaCysteine proteinasesEhCP112AutoactivationE-64 inhibition

1046-5928/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.pep.2008.09.006

* Corresponding author. Fax: +52 55 5747 3313.E-mail address: jortega@cinvestav.mx (J. Ortega-Ló

1 Abbreviations used: CPs, cysteine proteinases; ppEpre–pro cysteine proteinase 112; SDS–PAGE, sodium dgel electrophoresis; IPTG, isopropyl-b-D-thiogalactosideleucylamido(4-guanidino) butane; PMSF, phenylmethytosyl-L-lysine chloro methyl ketone hydro-chloride;acetic acid; DTT, dithiothreitol; PVDF, polyvinyldifluotrichloroacetic acid; BSA, bovine serum albumin; BCmethanol.

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complexinvolved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recom-binant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After arefolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or asparticproteinase inhibitors arrested this activation process. The activation step of the proenzyme followed bythe mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentallydetermined processing sites observed during EhCP112 activation lie close to processing sites of other cys-teine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined usinghemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited bythiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is aproteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step forits biochemical characterization and to carry out future protein structure–function studies.

� 2008 Elsevier Inc. All rights reserved.

Entamoeba histolytica is the causative agent of amoebiasis, aninfectious disease highly prevalent in developing countries. Thisparasite is characterized by invading and destroying human tis-sues, leading to potentially life-threatening diseases [1–3]. Severalvirulence factors are responsible for the pathogenicity of E. histoly-tica (i) molecules involved in adhesion of amoebae to target cells[2]; (ii) membrane-active factors involved in rapid killing of hostcells by exocytosis such as the ion channel-forming peptidesdubbed amoebapores [4,5], and (iii) secreted proteinases that de-grade extracellular matrix proteins and mediate tissue disintegra-tion [2,3,6–8]. Recently, more than 86 proteinases have beenidentified in the genome of E. histolytica. The vast majority of thembelong to the cysteine proteinase (CP)1 family with 50 members [9].

ll rights reserved.

pez).hCP112, Entamoeba histolyticaodecyl-sulfate polyacrylamide; E-64, trans-epoxysuccinyl L-

lsulfonyl fluoride; TLCK, Na-p-EDTA, ethylenediamine-tetra-ride; TB, terrific broth; TCA,

A, bicinchoninic acid; MeOH,

However, it has been reported that only few CPs are expressed incultured trophozoites [9–11]. Some CPs are involved in different as-pects of E. histolytica pathogenesis, such as destruction of host tis-sues or triggering an inflammatory response by the infectedindividuals [12,13]. Although, CPs are important pathogenicity fac-tors of this protozoan parasite [3,14], further characterization hasbeen hampered by difficulties in isolating and purifying these CPsfrom amoeba lysates and the inherent difficulties of their recombi-nant expression as active and stable enzymes [3,14–16].

EhCP112 is a cysteine proteinase that together with the adhesinEhADH112 form the EhCPADH complex involved in E. histolyticaadherence, phagocytosis and cytolysis [1,2,17]. Antibodies againstthe EhCPADH complex inhibited adherence, phagocytosis anddestruction of cell monolayers, by live trophozoites and greatly re-duced their ability to produce liver abscesses in hamsters [18]. Inaddition, a mixture of plasmids encoding Ehcp112 and Ehadh112genes inhibits liver abscess formation by virulent trophozoites inhamsters [19], making this complex an attractive vaccine candi-date against amoebiasis.

EhCP112 is a 446-amino acid, papain-like proteinase. Like allmembers of this family, it contains a signal peptide, a propeptideand a catalytic domain characterized by the catalytic triad (C, H,and N). EhCP112 also presents an ERFNIN motif, and a RGD se-

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quence, which may help to interact with cellular integrins [20]. Re-cently, three DNA fragments encoding the EhCP112 precursor(ppEhCP112), the proenzyme (pEhCP112) and the mature enzyme(EhCP112) were cloned into pTrcHisC and pET38b(+) vectors andexpressed with very low yields [3]. The ppEhCP112 and the matureenzyme were expressed as inactive enzymes, while the EhCP112proenzyme was expressed as an active proteinase [3]. This behav-ior is in contrast to other CPs from E. histolytica, like EhCP1, EhCP2,and EhCP5 that were recombinantly expressed as inactive enzymesfrom their proenzymes and needed to be refolded and activated[15,21]. In addition to the low protein yields observed during puri-fication, in the initial characterization of EhCP112, the recombinantproenzyme was very unstable due to its strong autoproteolyticactivity [3]. This behavior hampered the use of this construct asa suitable source of recombinant EhCP112 for further biochemicaland structural studies [3]. To overcome these problems and to beable to further characterize the EhCP112 proteinase, here we re-port the recombinant expression with high yield of the inactiveEhCP112 precursor, its activation under controlled conditions,and the biochemical characterization of the processed and matureproteinase.

Materials and methods

Cloning and expression of the ppEhCP112

The precursor ppEhCP112 gene was amplified by PCR using thedirect and reverse oligonucleotides: ppEhCP112-50 (50-AGGTCGGATCCATGAC AGCGATTGTTGTCGCT), ppEhCP112-30 (50-AGGCAAAGCTTTTAGATTGTATGATTGTAGAATTG), respectively, and using theplasmid pRSET A-ppEhCP112 as DNA template [2]. The primerscontained BamHI and HindIII restriction sites (bold) to allow direc-tional cloning of the amplified DNA into the prokaryotic expressionvector pQE80L (Qiagen). Transformed Escherichia coli M15 cellswith the pQE80L-ppEhcp112 construct were grown to inoculate500 ml of terrific broth (TB) (100 lg/ml ampicillin, 30 lg/ml kana-mycin) at 37 �C and 200 rpm; when cultures reached anOD600 = 0.8, the recombinant protein was induced by adding1 mM of IPTG for 4 h. Bacteria were resuspended in 300 mM NaCl,20 mM Tris–HCl pH 8.0 (1 ml buffer per gram of cells), supple-mented with lysozyme (1 mg/ml), incubated on ice for 30 min,and lysated by sonication. The lysate was centrifuged at 16,000gfor 30 min at 4 �C to isolate the soluble and insoluble fractions.

Purification of recombinant ppEhCP112 by nickel affinitychromatography

The insoluble protein fraction was washed twice with 5 ml of20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 2% Triton X-100, and2 M urea. Inclusion bodies were solubilized using 10 ml of bufferA (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 10 mM imidaz-ole, and 8 M urea). After 1 h incubation at room temperature, thesuspension was centrifuged at 16,000g for 30 min to remove insol-uble material. The recombinant ppEhCP112 was purified on a 1 mlprepacked column of Ni-Sepharose High Performance resin (GEHealthcare Science). The column was washed with 10 ml of deion-ized water and equilibrated using 5 ml of buffer A. Then the His-tagged protein was applied to the Ni-Sepharose column, washedwith 15 ml buffer A, and the affinity bound protein was eluted with5 ml of buffer A supplemented with 500 mM imidazole. 1 ml frac-tions were collected and analyzed by SDS–PAGE. Fractions contain-ing purified ppEhCP112 were pooled and stored at �20 �C. Theprotein concentration was determined using BCA (bicinchoninicacid) Protein Assay Kit (Pierce) following the manufacturer’s rec-ommendations using BSA (bovine serum albumin) as a standard.

Refolding and activation of the recombinant ppEhCP112

A volume of 2.5 ml of the purified and unfolded ppEhCP112 (1–2 mg/ml) was applied to a 1.45 � 8 cm PD10 Desalting Columnwith 8.3 ml of Sephadex G-25 (GE Healthcare Science) pre-equili-brated with refolding buffer (5 mM CaCl2, 0.02% SDS, 100 mMTris–HCl, pH 8.0). This simple purification step removes the ureaand allows the exchange of the protein to the refolding buffer.Therefore, it allows the protein to refold into its native conforma-tion. Processing of ppEhCP112 into an enzymatically active protein(EhCP112) was achieved by incubating 500 lg of the ppEhCP112 in1 ml refolding buffer supplemented with 10 mM DTT at 25 �C. Thisactivation protocol is similar to protocols successfully employedwith other recombinantly expressed CPs of E. histolytica, likeEhCP1, EhCP2 and EhCP5 [15,21,22].

To monitor the activation of ppEhCP112, 50 ll samples were ta-ken at 15, 20, 25, 30, 35, 40, and 45 min, and analyzed by SDS–PAGE and gelatin–SDS–PAGE. In addition, a spectrophotometricenzymatic assay using azocasein as substrate was performed tocorroborate proteolytic activity.

To determine whether the activation was an autocatalytic pro-cess, 20 lg of refolded ppEhCP112 in 500 ll of refolding bufferwere incubated at 37 �C for 10 min in the presence of 10 lM E-64, 1 mM PMSF, or 1 lM pepstatin A before the activation processwith DTT. These samples were analyzed by SDS–PAGE after 20 and35 min of activation.

Substrate gel electrophoresis assay

Proteinase activity was determined by a substrate–gel electro-phoresis using a 10% polyacrylamide gel copolymerized with0.1% gelatin as substrate (gelatin–SDS–PAGE or zymogram), as pre-viously reported [2,3]. Briefly, 20 ll samples were mixed with anequal volume of 2� Laemmli buffer, incubated 15 min at 30 �C,and 10 ll of each sample were loaded into a substrate–gel. Afterelectrophoresis, the gel was incubated for 1 h in 2.5% (v/v) TritonX-100 and for additional 30 min in 100 mM sodium acetate, pH5.2, 1% (v/v) Triton X-100, containing 20 mM DTT at 37 �C priorto staining with Coomassie brilliant blue. Clear bands were indica-tive of proteolytic activity.

Azocasein spectrophotometric assay

The proteolytic activity of EhCP112 was determined by a spec-trophotometric assay using azocasein as substrate, as previouslyreported [15,23]. Samples containing 1 lg of active enzyme or ac-tive enzyme-inhibitor were incubated in 500 ll of 0.1 M citricacid–0.2 M Na2HPO4, pH 7.0 buffer, containing 6.5 mg/ml of azoc-asein at 37 �C, and analyzed every minute for 10 min. Reactionswere stopped by the addition of one volume (500 ll) of 20%(w/v) trichloroacetic acid (TCA) and incubated at 4 �C for10 min. Samples were centrifuged at 10,000g for 15 min and theabsorbance of the supernatants was immediately determined at366 nm.

Processing site determination by N-terminal sequencing

After electrophoresis processed activation products weretransferred to a polyvinyldifluoride (PVDF) membrane, stainedwith 0.1% Coomassie brilliant blue in 40% methanol (MeOH),1% acetic acid, and destained with 50% MeOH. Bands of interestwere excised and stored at �20 �C. The sequence of the first tenN-terminal amino acids of the excised bands was obtained byEdman degradation (Protein Core Facility at Columbia University,USA).

Fig. 1. Expression and purification of the recombinant ppEhCP112. (A) SDS–PAGEanalysis of cell extracts from E. coli M15 harboring the pQE80L-ppEhcp112 constructbefore (lane 1) and after (lane 2) IPTG induction; analysis of soluble (lane 3) andinsoluble (lane 4) fractions after disruption of induced cells. (B) ppEhCP112solubilized from Inclusion bodies using 8 M urea (lane 1) and purified by nickelaffinity chromatography (lane 2). Purified ppEhCP112 was kept at �20 �C until itsactivation.

Table 1Purification and activation of the recombinant ppEhCP112a

Sample Total protein(mg)

Specific enzymeactivityb (U/mg)

Proteinyieldc (%)

Total cell lysate 1060 ND 100Soluble fraction 460 ND ND*

Insoluble fraction 600 ND �100Inclusion bodiesd 260 ND �85Flow throughe 190 ND ND*

Ni-affinity chromatography 50 ND �26Refolding and activationf 35 1450 �18

a A total of 9 g wet weight of cells from 500 ml culture were lysed and totalprotein was determined on each step by the BCA assay as indicated in Materials andmethods.

b Protein yield was estimated by densitometric analysis of protein bands in theSDS–PAGE gel, assuming that 100% of recombinant protein was in the insolublefraction.

c Enzymatic activity was measured using the azocasein assay as described inMaterials and methods.

d Inclusion bodies were solubilized in 8 M urea and loaded into the Ni-affinitycolumn.

e Unbound protein to the Ni-affinity column.f Purified and refolded precursor was activated by 35 min incubation at 25 �C in

100 mM Tris–HCl, pH 8.0, 5 mM CaCl2, 0.02% SDS and 10 mM DTT. ND, not detected;ND*, not determined.

28 L.I. Quintas-Granados et al. / Protein Expression and Purification 63 (2009) 26–32

Assessment of optimal pH and temperature for the EhCP112proteolytic activity

Proteolytic activity of EhCP112 was performed as previously de-scribed [3,15,23] using 1 lg of active EhCP112 in a 500 ll buffercontaining 3.1 mg/ml of hemoglobin or 6.5 mg/ml of azocasein assubstrates. The acid and basic conditions were assayed in 0.1 M cit-ric acid–0.2 M Na2HPO4 buffer pH 3.0–7.0; 0.2 M glycine–0.2 MNaOH buffer, pH 9.0 and 10.0, or 0.1 M Tris–HCl, pH 8.0. Since azoc-asein was insoluble at pH values less than 6.0, the active enzymewas incubated at 25 �C for 1 h at pH 3.0, 4.0, and 5.0; then, the pro-teolytic activity was measured using azocasein as described above.

To determine the optimum temperature of EhCP112, 1 lg ma-ture enzyme was incubated in 500 ll of buffer at different temper-atures (5, 25, 37, 40, 50, and 60 �C) using 6.5 mg/ml azocasein or3.1 mg/ml hemoglobin at optimum pH value for each substrate;then, EhCP112 proteolytic activity was measured spectrophoto-metrically as above.

Kinetic studies

Activity assays were performed in a reaction volume of 500 llusing 1 lg of active EhCP112 and different concentrations of azoc-asein (1.6, 3.2, 4.8, 6.5, and 8.0 mg/ml) or hemoglobin (0.6, 1.3, 1.8,2.5, and 3.1 mg/ml) in citric acid–Na2HPO4, pH 7.0 for azocaseinand pH 6.0 for hemoglobin. For azocasein, one unit of proteolyticactivity corresponds to the amount of enzyme releasing 1 lg azoc-asein per min under the assay conditions. The extinction coeffi-cient of azocasein e01%/366 nm = 3648 cm2 mg�1 was used forcalculating proteolytic activity [13]. For hemoglobin, one unit ofenzyme activity was defined as the amount of enzyme requiredto cause a unit increase in absorbance at 280 nm across a 1 cm pathlength under the conditions of the assay [23]. The initial velocities(V0) of the EhCP112-catalyzed reactions were calculated from theslopes of the linear initial progress curves. Km and Vmax values ofthe enzyme toward their corresponding substrates were deter-mined by nonlinear least squares regression fittings of the initialvelocity (V0) vs. substrate concentration ([S]) curves according tothe Michaelis–Menten equation using the program SigmaPlot 9.0.

Inhibition of proteolytic activity of EhCP112

For these studies, 20 lg of active EhCP112 were incubated in500 ll of 0.1 M citric acid–0.2 M Na2HPO4 buffer, pH 7.0 containingcysteine, serine, serine/cysteine, metallo, or aspartic proteinaseinhibitors at 37 �C for 10 min. The residual activity of 1 lg EhCP112was measured by azocasein (6.5 mg/ml) spectrophotometric assayand expressed as percentage of inhibition. The EhCP112 proteolyticactivity inhibition was also monitored by substrate–gel electro-phoresis. Briefly, 20 ll of inhibited samples were mixed with 2�Laemmli buffer, and incubated 15 min at 30 �C. Samples (10 ll)were loaded into a 10% SDS–PAGE copolymerized with 0.1% gelatinand processed as described above. The following inhibitors wereused: 10 lM E-64, 100 lM TLCK, 1 mM PMSF, 100 lM leupeptin,10 lM antipain, 1 mM N-ethylmaleimide, 1 lM pepstatin A,5 mM EDTA, and 100 lM chymostatin.

Results

Expression and purification of the precursor of EhCP112

The appearance of an overexpressed protein band of approxi-mately 52 kDa in the bacterial extracts after IPTG induction incomparison to cell extracts without IPTG clearly shows the expres-sion of the ppEhCP112 (Fig. 1A, lanes 1 and 2). In addition, the anal-ysis of the soluble and the insoluble fractions (lanes 3 and 4),

indicates that the ppEhCP112 was expressed mainly as an insolu-ble protein aggregate. This was corroborated by a Western blotanalysis with an anti-His monoclonal antibody, which only recog-nized the recombinant precursor in the induced bacterial extractand insoluble fraction; but neither in the soluble fraction nor innon-induced cells (data not shown). The ppEhCP112 was solubi-lized from inclusion bodies using 8 M urea (Fig. 1B, lane 1) andpurified by Ni-affinity chromatography, under denaturing condi-tions (lane 2). A yield of 50 mg of the purified ppEhCP112 was ob-tained per 500 ml of cell culture (Table 1) and stored at �20 �C in8 M urea at a concentration of approximately 1 mg/ml for furtheractivation.

Refolding and activation of EhCP112

Thus, in order to activate the EhCP112 precursor (52 kDa), thepurified recombinant ppEhCP112 was first refolded by exchangingthe denatured protein to a refolding buffer without urea by one gelfiltration step on a PD10 column. After the addition of 10 mM DTT

L.I. Quintas-Granados et al. / Protein Expression and Purification 63 (2009) 26–32 29

to the refolded polypeptide, its activation kinetics for 45 min wasfollowed by a 15% SDS–PAGE (Fig. 2A). As expected, at the begin-ning of incubation, only the ppEhCP112 band was observed (lane1). This protein band progressively decreased through time (lanes2–4) and disappeared at 30 min of the activation process (lane5). In this kinetic study, an additional band of 47 kDa was detectedafter 15 min of activation (lane 2) and disappeared after 35 min(lane 6). A band of 35.5 kDa was detected after 20 min (lane 3)and throughout the experiment (lanes 4–8) with a maximumappearance at 35 min (lane 6). Also, a protein band with apparentmolecular weight of 16 kDa was detected after 20 min andthroughout the experiment (lanes 3–8). The 47 kDa band may cor-respond to the cleavage of the signal peptide of the enzymaticallyinactive precursor giving arise to the proenzyme. The 47 kDa bandwas processed to a 35.5 kDa band that may correspond to theenzymatically active mature enzyme. The reduction in intensityof the 35.5 kDa band may indicate the autoproteolysis of the acti-vated enzyme.

To corroborate the autoactivation process of the enzymaticallyinactive precursor, samples subjected to the activation processwere also analyzed by substrate gel electrophoresis using gelatinas substrate (Fig. 2B). No activity was detected at the time pointsin which only the ppEhCP112 of 52 kDa and the 47 kDa polypep-tides were present (lanes 1 and 2), indicating that this 47 kDaband corresponds to an inactive form of the EhCP112 peptidase.Bands with proteolytic activity were observed only at the positioncorresponding to the 35.5 kDa band (lanes 3–8), indicating thatthis band corresponds to the active EhCP112 proteinase. The max-imum activity was observed at 35 min (lane 6), which correlates

Fig. 2. Activation of the ppEhCP112. (A) 15% SDS–PAGE analysis depicting the mobility ofincubation times t = 0 (lane 1), 15 min (lane 2), 20 min (lane 3), 25 min (lane 4), 30 min (lthe gel indicate the recombinant precursor (52 kDa), the proenzyme (47 kDa), matureEhCP112. (B) Zymogram on a 10% polyacrylamide–gelatin gel with the same samples loaprotein bands of 35.5 kDa. (C) Analysis of proteolytic activity during activation of EhCP11azocasein, as a substrate. The specific activity was estimated using the initial amount ofautoprocessing of EhCP112 precursor in the presence of DTT. Twenty microgram of ppEproteinases inhibitors: 10 lM E-64 (lane 1, E-64), 1 mM PMSF (lane 2, PMSF); 1 lM pepstthe right side of the gel indicate the recombinant precursor (52 kDa) and the mature (3

with the maximum intensity of the 35.5 kDa band observed onthe SDS–PAGE (Fig. 2A, lane 6). These data show that inactiveppEhCP112, under this incubation conditions, was autoprocessedto generate the mature active enzyme. It is noteworthy to men-tion that intensity of the 35.5 kDa band (Fig. 2A, lane 6) was pro-portional to the enzyme activity observed on the zymogram(Fig. 2B, lane 6) and the one determined by the azocasein assay(Fig. 2C). In both cases, the highest proteolytic activity was ob-served at 35 min with a steady decrease thereafter. Interestingly,reproducible refolding and activation results were obtained withthe recombinant ppEhCp112, even after 3-month storage in 8 Murea (data not shown).

To demonstrate that the ppEhCP112 is autoactivated underreductive conditions, the ppEhCP112 was preincubated for10 min with several proteinase inhibitors followed by incubationwith DTT for 35 min (Fig. 2D). At 20 min incubation the 52, 47,and 35.5 kDa protein bands were observed in samples with serine(PMSF), aspartic (Pep A), or without inhibitor (data not shown). At35 min incubation, the activation of the precursor was not ob-served when the specific thiol proteinase inhibitor E-64 was in-cluded in the incubation mixture (lane 1). Nevertheless, whenserine (PMSF) (lane 2) and aspartic (Pep A) (lane 3) proteinaseinhibitors were used, the 35.5 kDa protein band of the mature en-zyme was observed with the same intensity as in the controlexperiment without inhibitor (lane 4). The arrest of the activationof EhCP112 precursor by the specific thiol proteinase inhibitor E-64, but not by serine (PMSF) or aspartic (PepA) proteinase inhibi-tors indicates that the ppEhCP112 in vitro activation process underreductive conditions is indeed an autocatalytic reaction.

the products from autoprocessing of recombinant ppEhCP112 using DTT at differentane 5), 35 min (lane 6), 40 min (lane 7), and 45 min (lane 8). Lines in the right side of

(35.5 kDa), and the 13.2 kDa abnormally run prosequence fragment (16 kDa) ofded in (A). The white bands indicate proteolytic degradation of gelatin only by the

2 precursor. Samples from lanes 1–8 in (A) were also assayed for their activity usingppEhCP112 in the activation assay. (D) 10% SDS–PAGE analysis after 35 min of the

hCP112 were incubated at 37 �C for 10 min in 500 ll of refolding buffer containingatin A (lane 3, PepA); or without proteinase inhibitor, as a control (lane 4, C). Lines in5.5 kDa). Gels (A, B, and D) were stained with Coomassie brilliant blue.

30 L.I. Quintas-Granados et al. / Protein Expression and Purification 63 (2009) 26–32

Determination of the processing sites involved in EhCP112 maturation

Although the putative processing sites of inactive ppEhCP112,to form a mature EhCP112 enzyme have been deduced from itsamino acid sequence by an in silico analysis [2,3,10]. The preciseprocessing sites of EhCP112 have not been experimentally deter-mined. N-terminal sequencing results of the first 10 amino acidsof the 47 and 35.5 kDa protein bands indicate that the 47 kDa pro-tein band corresponds to a 427-amino acid proenzyme starting atposition Ile20, which lacks the 19-amino acid signal peptide. The35.5 kDa protein band corresponds to the mature enzyme with315 residues starting at Leu132. The 16 kDa protein band was alsoN-terminal sequenced (Fig. 2A) and found to have the same start-ing position that the 427-amino acid proenzyme (Ile20). This find-ing indicates that the 16 kDa protein band corresponds to the 112-amino acid profragment with the expected molecular weight of13.2 kDa, which on the SDS gel ( Fig. 2A) run abnormally as a16 kDa protein band.

The schematic organization of the processing sites involved inEhCP112 maturation is depicted in Fig. 3. The N-terminal sequenceof processing fragments and the inhibition of activation resultsindicate that the ppEhCP112 (52.1 kDa) activation process underreductive condition is an autocatalytic sequential reaction; firstthe 19-amino acid presequence is removed to generate the proen-zyme (47 kDa), followed by the 112-amino acid profragment(13.2 kDa) to generate the 315-amino acid mature enzyme(35.5 kDa).

Optimal pH and temperature conditions for the EhCP112 proteolyticactivity

Establishing the interval and optimal pH and temperature con-ditions for the enzymatic activity of the mature EhCP112 protein-ase is essential to obtain detailed kinetic studies, which may be

Fig. 3. Schematic representation of the processing profile of recombinantppEhCP112 activation. Schematic representation of the ppEhCP112 autoprocessingbased on the N-terminal sequence of the 47, 35.5, and 16 kDa protein bands(Fig. 2A). kDa values correspond to the estimated molecular weight from the aminoacid sequence of each protein fragment. The 52.1 kDa recombinant ppEhCP112includes the 446-amino acid of the full length EhCP112 precursor and the His-tag.The M indicates the methionine at position 1. I at position 20 and L at position 132indicate the N-terminal of the profragment and mature enzyme, respectively. The(*) C, H, and N on the mature fragment indicate the catalytic residues and RGDindicates the integrin-binding sequence. The 10-amino acid in the N-terminal of the16 kDa protein band were identical to the10-amino acid in the N-terminal of the47 kDa protein band (Fig. 2A). Thus, the 16 kDa protein band corresponds to the112-amino acid profragment with an expected size of 13.2 kDa, but with anabnormal migration on the SDS–PAGE.

correlated to the in vivo conditions. Fig. 4A and B show an analysisof the EhCP112 proteolytic activity, as a function of pH and tem-perature, respectively using azocasein and hemoglobin as sub-strates. The EhCP112 proteinase was enzymatically active at a pHinterval from 3.0 to 10.0 for both substrates. The highest activitywas observed at pH 7.0 for azocasein and pH 6.0 for hemoglobin.The EhCP112 was enzymatically active from 5 to 60 �C with a max-imum activity for both substrates at 37 �C.

Kinetics and inhibition studies of EhCP112

Kinetics parameters of the mature EhCP112 enzyme weredetermined using azocasein and hemoglobin as substrates(Fig. 4C). Initial velocity (V0) vs. substrate concentration data fitted

Fig. 4. Biochemical characterization of the mature recombinant EhCP112. (A) pH-activity profiles for the mature EhCP112 on azocasein (s) and hemoglobin (d). Thetested pHs were at the interval of 3 to 10 at 37 �C using a constant ionic strengthbuffer of citric acid–Na2HPO4 or glycine–NaOH. (B) Temperature–activity profilesfor the mature EhCP112 on azocasein (s) and hemoglobin (d) at pH 7.0 and 6.0,respectively, from 4 to 60 �C. (C) Initial hydrolysis rate (U/mg) at differentconcentration (mg/ml) of azocasein (s) at pH 7.0 and hemoglobin (d) at pH 6.0of mature EhCP112 at 37 �C. Lines represent the best fit of the experimental data tothe Michaelis–Menten equation by a least squares nonlinear regression. Verticalbars indicate the standard error.

Table 2Kinetic constants for the recombinant EhCP112

Substrate Vmax (U/mg) Km (mg/ml) kcat (seg�1) kcat/km (seg�1 mg�1 ml)

Azocasein 1450 1.5 48 32Hemoglobin 1600 0.4 54 135

Hydrolysis was performed at 37 �C and pH 7.0 for azocasein or pH 6.0 for hemo-globin. The enzyme was activated by 35 min incubation at 25 �C in 100 mM Tris–HCl, pH 8.0, 5 mM CaCl2, 0.02% SDS and 10 mM DTT.

Fig. 5. Effect of proteinase inhibitors on EhCP112 activity. Mature EhCP112 wasincubated for 10 min with various proteinase inhibitors as indicated in Materialsand methods. (A) To detect residual proteolytic activity, samples were separated on10% gelatin–SDS–PAGE. Gels were processed as described under Materials andmethods and stained with Coomassie brilliant blue. Clear bands represent region ofactive EhCP112. (B) Samples were also assayed for residual activity by the azocaseinspectrophotometric assay. The percentage of inhibition was estimated using theproteolytic activity of EhCP112 (without proteinase inhibitors) as 0% of inhibition.

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to a Michaelis–Menten equation. The Vmax and kcat values forhemoglobin were slightly higher than those observed for azoca-sein; however, the Km for hemoglobin was three times lower. Thus,the catalytic efficiency of the mature EhCP112 enzyme for hemo-globin is more than four time higher than for azocasein (Table 2).

To further demonstrate that EhCP112 is indeed a cysteine pro-teinase, the mature enzyme was incubated with several knownproteinase inhibitors. The proteolytic activity of EhCP112 waspoorly inhibited by specific serine, metallo, or aspartic proteinaseinhibitors (PMSF, EDTA, and Pepstatin-A, respectively); moderately(N-ethylmaleimide) and strongly (leupeptin and antipain) inhib-ited by the non specific CP inhibitors. The thiol inhibitors chymo-statin, TLCK, and E-64 completely inhibited the EhCP112proteolytic activity (Fig. 5).

Discussion

Entamoeba histolytica harbors at least 50 different CPs of theclan CA [9]. Since only few of these genes had been cloned andcharacterized, the vast majority of the knowledge of CPs activityin amoeba is based on the analysis of native CPs obtained from cul-tured trophozoites [3,10,15,16,21]. EhCP112, as other cysteine pro-teinases, is a potential candidate for new drugs against the parasiteinvasion. However, structural and functional studies of these pro-teinases had been hampered because of the low yields of activeproteinases using recombinant expression systems [3,15,21]. In aprevious work, the precursor, the proenzyme, and mature con-structs of EhCP112 were expressed in E. coli with very low expres-sion yields [3]. The recombinant precursor and mature EhCP112proteins were inactive. However, the recombinant proenzyme dis-played proteinase activity, in addition to a strong autocatalytic

activity that affected it usefulness for further studies [3]. The enzy-matic activity of the EhCP112 proenzyme contrasts to the catalyticinactivity of the recombinant pro-enzymes, EhCP1, EhCP2, andEhCP5 that needed to be activated [15,21,22]. As a consequenceof those results, we developed a strategy to maximize proteinexpression of the inactive EhCP112 precursor in inclusion bodies.We subcloned ppEhCP112 in a pQE80L vector, which contains anextra copy of the lac I repressor, giving a tighter expression controlthan pTrcHisC and pET38b plasmids, which possibly helps to avoidtoxicity by a population of active proteinases. The ppEhCP112 wasexpressed at high levels in bacteria as a 52 kDa polypeptide withan improvement in the expression levels previously obtained(Fig. 1A and data not shown) [2,3]. Although the ppEhCP112 pro-tein was present as inclusion bodies, we were able to purify andsolubilize them at yields of 50 mg per 500 ml of culture (Table1). This yield is considerably superior (�50-fold) than yields ob-tained previously with this and other cysteine proteinases of E. his-tolytica [3,15].

Previous studies have shown that recombinant cysteine pro-teinases are able to undergo maturation by the removal of theirprosequence by autocatalytic cleavage [15,21,24]. Recent studieshave shown the autoactivation of other cysteine proteinases fromE. histolytica by combining DTT and detergents [15,16,21,22]. Here,we present a similar activation-treatment for the recombinantppEhCP112 (Fig. 2A). The process is characterized by progressivedisappearance of the recombinant ppEhCP112 (52 kDa band)through time, accompanied by an increase in the amount of activeEhCP112 (35.5 kDa band), the appearance of an intermediate pro-enzyme of 47 kDa, and a stable 16 kDa band. N-terminal sequenceof the 16 kDa protein band indicates that this protein fragmentcorresponds to the 112-amino acid (13.2 kDa) profragment ofEhCP112 with an abnormal migration in the SDS–PAGE (Fig. 2A).

In our hands this activation process is able to produce after35 min of incubation a single band of 35.5 kDa with proteolyticactivity. The yield of the protein band of the mature EhCP112 is18% and gives arise to 35 mg per 500 ml of cell culture (Table 1).We corroborate that this band is the mature active enzyme by azymogram and azocasein activity assays. For instance, in these as-says after 15 min of activation no detectable amount of matureproteinase is observed by SDS–PAGE (Fig. 2A lane 2) and corre-spondingly no proteinase activity is present in the zymogramand the activity assay (Fig. 2B lane 2 and Fig. 2C). We demonstratedthat the activation process is indeed an autoactivation process,since this process was inhibited by the action of the specific thiolproteinase inhibitor E-64, but not by serine and aspartic proteinaseinhibitors (PMSF and Pep A) (Fig. 2D).

We also determined the processing sites of the autoactivationproducts. EhCP112 consist of a hydrophobic pre-sequence of 19-amino acid, a prodomain of 112-amino acid, and a catalytic domain(mature enzyme) of 315-amino acid, according to the N-terminalsequence of autoactivation products. The processing sites involvedin EhCP112 maturation lie close to the processing sites observed inrecombinant expressed CPs of other parasites and is slightly differ-ent with the previously maturation sites determined by in silicoanalysis (Fig. 3) [3,25,26].

In addition, the recombinant active EhCP112 enzyme was ableto degrade hemoglobin and azocasein. The enzyme showed amarked preference for hemoglobin, as assessed by the catalyticefficiency on both substrates (Table 2). These results are expected,since hemoglobin is a natural substrate that E. histolytica encoun-ters during infection, suggesting that EhCP112 is involved in hemo-globin degradation in vivo.

Many parasitic CPs are active and remain stable at neutral orslightly alkaline pH [8,25]. The optimal pH for proteinase activityof EhCP112 was 6.0 and 7.0 for hemoglobin and azocasein, respec-tively. Nevertheless, it is important to mention that EhCP112 was

32 L.I. Quintas-Granados et al. / Protein Expression and Purification 63 (2009) 26–32

found to be enzymatically active at the pH range 3.0–10.0 (Fig. 4A),similar to the pH range previously described for the recombinantproenzyme EhCP112 using polyacrylamide–gelatin gels [3].EhCP112 has high homology with papain-like CPs. This proteinasefamily is considered to be unstable at neutral pH. However, onlysome are relatively unstable (cathepsins L, B, H, K V, and F);whereas, others (cathepsin S, cruzipain) were found to be extre-mely stable, possibly accounting for their role outside lysosomes[27,28]. The EhCP112 showed high activity at physiological pHand 37 �C (Fig. 4B), which is consistent with the fact that the EhC-PADH complex is secreted during infection [2,3]. EhCP112 might beactive at the extracellular environmental conditions. How theEhCP112 is activated and regulated in vivo remains to be deter-mined. It is possible that the EhCP112 precursor interacts withthe adhesin EhADH to form the EhCPADH complex and this associ-ation prevents the proteinase activation before its translocation tothe surface of E. histolyica. Also, it is possible that one of the E. his-tolytica CP inhibitors, EhICP1 or EhICP2, might participate in pro-cessing and activation of EhCP112, as suggested for other CPs [21].

The identity of the EhCP112 as a thiol proteinase of clan CA wasfurther confirmed by inhibitor studies. Reagents known to specifi-cally inhibit serine, aspartic, or metallo proteinases did not influ-ence EhCP112 activity (Fig. 5). In contrast, enzymatic activity ofEhCP112 was greatly reduced or completely blocked with all CPinhibitors tested such as E-64, leupeptin, chymostatin, antipainand TLCK (Fig. 5). Members of the clan CA are characterized bytheir sensitivity to the general cysteine proteinase inhibitor E-64[8]. Thus based on these results, the EhCP112 is classified as a pa-pain-like cysteine proteinase.

In conclusion, the work outlined here describes a technique toprepare considerable amounts of purified ppEhCP112 and an acti-vation protocol to convert 70% of precursor into an enzymaticallyactive, recombinant cysteine proteinase of E. histolytica. In thiswork we have succeeded in the molecular cloning of ppEhcp112and its expression using the E. coli M15 strain, obtaining a largeamount of purified His-tagged EhCP112 activable precursor. In35 min about 70% of ppEhCP112 was autoprocessed into the 315-amino acid active enzyme. The catalytic properties of EhCP112are consistent with the in vivo role of EhCP112 in pathogenesis.

Acknowledgments

This work was partially supported by a Grant 40387-Z (JOL)CONACYT and a TWAS research Grant (LGB). LIQG is a recipientof a scholarship from CONACYT. Thanks to Dr. Guillermina Gar-cia-Rivera, Victor Hugo Rodriguez Vargas, and José Luis MoralesRomero for their technical assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.pep.2008.09.006.

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