Entamoeba histolytica: Biochemical characterization of a protein disulfide isomerase

Experimental Parasitology 128 (2011) 76–81

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Entamoeba histolytica: Biochemical characterization of a protein disulfide isomerase Marco A. Ramos ⇑, Rosa E. Mares, Paloma D. Magaña, Israel D. Rivas, Samuel G. Meléndez-López Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Tecnológico 14418, Tijuana, Baja California 22390, Mexico

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Article history: Received 4 October 2010 Received in revised form 5 February 2011 Accepted 8 February 2011 Available online 12 February 2011 Keywords: Entamoeba histolytica Protein disulfide isomerase Oxidoreductase Biochemical characterization

a b s t r a c t Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The oxidation and correct formation of disulfide bonds are important biochemical modifications of many proteins (Wong et al., 2011). Early observations showed that disulfide bond formation proceeds much faster in vivo than in vitro, suggesting the existence of a catalyst for protein oxidative folding in living cells (Roth and Koshland, 1981; Frand et al., 2000). Enzymes from the protein disulfide isomerase family (PDI, EC 5.3.4.1) are eukaryotic oxidoreductases that catalyze the oxidation, reduction and isomerization of disulfide bonds in nascent polypeptides (Gilbert, 1997). The subcellular localization and its function suggest that PDI plays a key role in the folding of proteins delivered to the secretory pathway (Freedman, 1989). For the protozoan parasite Entamoeba histolytica, the causative agent of human amebiasis (WHO, 1997), the accurate formation of disulfide bonds is an important modification required for the correct folding of proteins, including those involved in adhesion and destruction of human tissues (Mann, 2000; Hecht et al., 2004). Thus, the identification and characterization of enzymes that play key roles in protein folding and secretion will be essential for further understanding of the cell biology of this protozoan parasite (Laughlin and Temesvari, 2005).

Abbreviations: Eh, Entamoeba histolytica; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; LZM, lysozyme. ⇑ Corresponding author. Address: Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Tecnológico 14418, Mesa de Otay, Tijuana, Baja California 22390, Mexico. Fax: +52 664 6822790. E-mail address: [email protected] (M.A. Ramos). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.02.009

An entamoebal PDI enzyme (EhPDI) that exhibits oxidative refolding activity in vivo as well as oxidative and reductive activities in vitro has been identified (Ramos et al., 2005; Mares et al., 2009). Structurally, EhPDI shares domain architecture with the PDI P5/class-2 homologues (Ramos et al., 2005), featuring two active thioredoxin domains and a D-domain (also known as Erp29c domain). Here, we report the enzymatic characterization as well as the effects of the inhibitor bacitracin and pH on the activity of EhPDI. Furthermore, the question of whether EhPDI could act as chaperone-like protein was also addressed. These results represent a primer to understand how this enzyme participates in protein folding and, consequently, in the virulence and lifestyle of E. histolytica. 2. Materials and methods 2.1. Reagents Chemicals and biochemicals were from Sigma–Aldrich and New England Biolabs, otherwise mentioned in the text. 2.2. Plasmid construct The cDNA encoding the mature polypeptide of EhPDI was amplified by PCR using the plasmid pBPelB:EhPDI as template (Ramos et al., 2005) and the synthetic oligonucleotides 50 -cggatccGCTGA TGTAGTATCATTAAATC-30 and 50 -gggaagcttaGAAAACTTCAAGTACA TT-30 as sense and anti-sense primers, respectively. The PCR product was digested with BamHI and HindIII endonucleases and then subcloned in-frame into the plasmid pQE30 (Qiagen). The

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recombinant plasmid (pQHPDI) was initially characterized by endonuclease digestion and then by DNA sequencing.

Windows (GraphPad www.graphpad.com).

2.3. Expression and purification of recombinant EhPDI

2.4.5. Effect of pH The effect of pH on the reductase and oxidase activities of recombinant EhPDI was determined by using an universal buffer system (Ellis, 1961). The reductase activity was carried out as previously described (Holmgren, 1979); while the oxidase activity was assayed using denatured and reduced ribonuclease A (drRNAse) as substrate following a standard spectrophotometric method (Lyles and Gilbert, 1991; Mares et al., 2009).

The recombinant EhPDI protein was expressed in Escherichia coli XL1-Blue MRF’ harboring the plasmid pQHPDI and purified form bacterial lysates as recommended by the manufacturer (Qiagen). Briefly, after 4 h of induction with 0.1 mM of IPTG, the bacterial cells were harvested and lysed under denaturing conditions. The soluble fraction was slowly loaded to a Ni–NTA-agarose column and the bound-protein was on-column refolded by exchanging the buffers to native-oxidative conditions. Subsequently, the protein was eluted with buffer containing imidazole (250 mM). The fractions containing significant amounts of protein were pooled and loaded to a PD10 column (Amersham). Finally, the recombinant EhPDI was eluted using 20 mM Tris–HCl buffer (pH 8.0). Protein concentration was determined by the Bradford colorimetric assay, using BSA as standard, or by UV spectrophotometry at A280, using the calculated absorptivity coefficient, 0.1% = 0.856 (Pace et al., 1995). 2.4. Oxidoreductase activity 2.4.1. Reductase assay The disulfide reduction of bovine insulin catalyzed by the recombinant EhPDI enzyme was assayed according to a standard turbidity method (Holmgren, 1979). Different concentrations of insulin were added to a buffer containing 0.054 mg/mL of recombinant EhPDI. The reaction was followed by recording the absorbance at 650 nm (A650) for 60 min. 2.4.2. Oxidase and isomerase assays The oxidative refolding of denatured-reduced lysozyme (drLZM) and disulfide isomerization of scrambled lysozyme (scLZM) were assayed following a reported protocol (Katiyar et al., 2001). Reactivation of lysozyme was achieved by diluting different concentrations of drLZM, or scLZM, into a buffer containing 0.41 mg/mL of recombinant EhPDI. The reactivation assays were followed by sampling the reaction for 60 min. The amount of active lysozyme recovered by EhPDI oxidase or isomerase activity was determined by performing a lytic activity assay (Moreira-Ludewig and Healy, 1992; Katiyar et al., 2001). Both, oxidase and isomerase, activities were expressed as units of lysozyme per min.

Software,

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2.5. Chaperone-like activity The relative chaperone-like activity of recombinant EhPDI was tested by measuring the ability of this protein to prevent the aggregation of insulin (Scheibel et al., 1998). Initially, insulin was preincubated at 43 °C with different concentrations of EhPDI. Then, chemical-reduction was started by the addition of DTT (20 mM final). The reaction was followed by recording the A650 for 50 min. The relative chaperone-like activity of EhPDI was determined as the percentage of protection against aggregation, using the formula: % protection = [(A0  A)/A0]  100, where A0 and A represent the apparent saturation absorbance (here, usually after 30 min) in the absence and presence of EhPDI, respectively (Spinozzi et al., 2006). 3. Results We have previously identified an E. histolytica PDI homologue that exhibits both oxidase and reductase activities (Ramos et al., 2005; Mares et al., 2009); similarly, we have found that these were comparable to those observed for bacterial thioredoxin and bovine PDI (Mares et al., 2009). Here, we report comprehensive studies regarding its biochemical features, which will be a key to understand the role of EhPDI in disulfide bond formation and protein folding. Initially, the oxidoreductase activity assays were performed at pH 7.0, similar to the pH value determined for the ER compartment of typical cells (Kim et al., 1998; Demaurex, 2002). Then, the activity constraint by pH and the inhibitory effect by bacitracin were also analyzed. Additionally, we tested whether PDI behaves as a chaperone-like protein. 3.1. EhPDI has low reductase activity

2.4.3. Inhibition assay The effect of the antibiotic bacitracin, a well-known PDI inhibitor (Mandel et al., 1993), on the oxidoreductase activities of the recombinant EhPDI was determined using different concentrations of the inhibitor. After 30 min of enzyme/inhibitor interaction, the residual oxidoreductase activity was measured as previously described (Holmgren, 1979; Moreira-Ludewig and Healy, 1992; Katiyar et al., 2001). For the reductase activity, 131 lM of insulin was used as substrate; while, 20 lm of drLZM or 8 lM of scLZM were used as substrate for the oxidase or isomerase activity, respectively. 2.4.4. Enzyme and inhibition parameters Km and Vmax values were determined by linear least squares regression fitting of the reciprocal values of the initial velocity (1/V0) versus the substrate concentration (1/[S]) according to the Lineweaver–Burk (L–B) equation. IC50 values were determined by non-linear least squares regression of the relative oxidoreductase activity versus the logarithm of the inhibitor concentration according to a dose-dependent variable–slope fitting curve. These parameters were calculated using GraphPad Prism version 4.0 for

The disulfide bond reduction of insulin, followed by turbidity measured at A650, was used to analyze the reductase activity. Initially, we found that the precipitation of the free B-chain was stimulated by the increasing concentration of recombinant EhPDI (data not shown). Then, we studied recombinant EhPDI as an enzymatically active reductase. Our findings show that the initial velocity increases with increasing insulin concentration (Fig. 1). We found an apparent Km value of 2.026 mM. This is particularly high, compared with the value observed for the bacterial thioredoxin (0.011 mM), a well-known reductase (Holmgren, 1979). Furthermore, the structural motif CXPC, located in active sites of common disulfide reductants, such as the human ERdj5 (Hebert and Molinari, 2007; Ushioda et al., 2008), was not found in the thioredoxin domains of EhPDI; in contrast, the CXHC motif was found, which is prevalent in efficient thiol/disulfide oxidants (Ramos et al., 2008). Even though we consider that the reductase activity is not the major function of EhPDI, there is always the possibility that the recombinant enzyme behaves differently than the native enzyme; since, it could probably be linked to another cellular function, such as protein unfolding/degradation (Molinari et al., 2002).

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Fig. 1. Reductase activity of recombinant EhPDI. Different micromolar (lM) concentrations of bovine insulin were added to the reaction buffer (pH 7.0): 0 (open square), 44 (closed square), 87 (open circle), 131 (closed circle), 174 (open diamond), 262 (closed diamond). After 15 min of reaction, the insulin disulfide reduction was evaluated by measuring the A650. Each point represents the mean of three independent experiments (bar, standard error of the mean). The small upper left-sided panel represents the double-reciprocal Lineweaver–Burk plot.

3.2. EhPDI has efficient oxidase and isomerase activities Regarding the studies on the oxidase and isomerase activity of EhPDI, two variants of lysozyme were used as substrate, reduceddenatured (drLZM) or scrambled (scLZM). Lysozyme is a protein known to require four correct disulfide bonds for proper folding and enzymatic activity; therefore, it is a suitable substrate to analyze the functional activities of disulfide oxidoreductases (Katiyar et al., 2001). Initially, we found that the conversion of the drLZM or scLZM to native LZM (nLZM) was efficiently catalyzed by increasing concentrations of recombinant EhPDI (data not shown). Subsequently, we studied the recombinant EhPDI as an enzymatically active oxidase or isomerase. Our findings show that the initial velocity increases with increasing substrate concentration. As shown in Figs. 2 and 3, the rate of refolding is dependent on the concentration of the substrate (drLZM or scLZM); thus increasing the units of active nLZM. From the L–B plots, we calculated apparent Km values of 25.3 and 11.6 lM for the oxidase and isomerase activity, respectively; whereas the corresponding Vmax values were 8.7 and 11.9 units of nLZM recovered per min per mg of EhPDI. Despite the slight difference between the Km values, the micromolar order and the highly similar enzymatic rate suggest that EhPDI acts mainly as oxidase/isomerase. 3.3. Bacitracin inhibits EhPDI The inhibitory effect of the antibiotic bacitracin, a well-known inhibitor of PDI enzymes (Roth, 1981; Mizunaga et al., 1990; Mandel et al., 1993), was evaluated on the oxidoreductase activities of recombinant EhPDI. Typical inhibition profiles were observed (Fig. 4) for the reductase, oxidase and isomerase activities, with apparent IC50 values of 1.137, 0.172 and 0.334 mM, respectively. The IC50 value for the reductase activity is almost eight times higher than that reported for mammalian PDI (0.15 mM) (Smith et al., 2004). In contrast, the IC50 values for the oxidase and isomerase activities are highly similar to those observed for the leishmanial PDI (0.10 mM) (Ben Achour et al., 2002), yeast and mammalian PDI (0.20–0.30 mM) (Mizunaga et al., 1990). These results suggest that bacitracin can be a good inhibitor for the oxidase/isomerase activities of EhPDI.

Fig. 2. Oxidase activity of recombinant EhPDI. Different micromolar (lM) concentrations of denatured-reduced hen lysozyme (drLZM) were added to the reaction buffer (pH 7.0): 0 (open square), 5 (closed square), 10 (open circle), 15 (closed circle), 20 (open diamond), 25 (closed diamond), 30 (open triangle). The oxidative refolding of drLZM was evaluated by measuring recovered lytic activity of lysozyme (defined as the change of 0.01 units of A450 per min). Each point represents the mean of three independent experiments (bar, standard error of the mean). The small upper left-sided panel represents the double-reciprocal Lineweaver–Burk plot.

Fig. 3. Isomerase activity of recombinant EhPDI. Different micromolar (lM) concentrations of scrambled reoxidized hen lysozyme (scLZM) were added to the reaction buffer (pH 7.0): 0 (open square), 2 (closed square), 4 (open circle), 6 (closed circle), 8 (open diamond), 10 (closed diamond). The disulfide reshuffling of scLZM was evaluated by measuring the recovered lytic activity of lysozyme (defined as the change of 0.01 units of A450 per min). Each point represents the mean of three independent experiments (bar, standard error of the mean). The small upper leftsided panel represents the double-reciprocal Lineweaver–Burk plot.

3.4. EhPDI activity is restricted by pH The reductase and oxidase activities of EhPDI were evaluated at different pH values (5–11), in a buffer containing 50 mM of each citrate, phosphate, tris and carbonate. The reductase activity was optimal at a pH value of 6.5 (almost 90% of maximal activity is retained between pH 6.0 and 7.0), while oxidase activity has its optimum at the pH value of 8.0 (almost 90% of maximal activity is retained between pH 7.0 and 8.0) (Fig. 5). As expected, the oxido-

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Fig. 4. Inhibition of the oxidoreductase activities of recombinant EhPDI. Different millimolar (mM) concentrations of bacitracin were incubated with the enzyme, 30 min before the assay. Residual activity was determined as described in materials and methods: reductase (open circle), oxidase (open diamond), and isomerase (open square). Each point represents the mean of three independent experiments (bar, standard error of the mean).

Fig. 6. Chaperone-like activity of recombinant EhPDI. Different micromolar (lM) concentrations of EhPDI were incubated with bovine insulin, at 43 °C for 30 min, before the assay: 2.5 (closed square), 5.0 (open square), 7.5 (closed circle), and 10 (open circle). The ability of EhPDI to prevent the aggregation of insulin was determined as described in materials and methods. Each point represents the mean of three independent experiments (bar, standard error of the mean).

reductase activities of recombinant EhPDI are pH-dependent, e.g., the reductase activity is favored by acidic pH values and oxidase activity by alkaline pH values. These findings suggest that EhPDI could be a multifunctional enzyme that switches from one oxidoreductive activity to another in response to environmental pH changes.

aggregation (Wang and Tsou, 1993; Spinozzi et al., 2006). PDI enzymes are multifunctional proteins that could display chaperone activity (Wang and Tsou, 1993); however, contrasting results were observed in assays using different protein-substrates (lacking disulfide bonds), such as citrate synthase (Katiyar et al., 2001) and rhodanese (Mouray et al., 2007). So, we decided to approach this function by using a simple chaperone-like activity assay (Scheibel et al., 1998). As observed in Fig. 6, protein aggregation is diminished by the increasing concentration of recombinant EhPDI, as consequence of its chaperone-like activity. It is worth mentioning that to demonstrate EhPDI as bona fide chaperone it must display ability to stabilize newly synthesized polypeptides during folding, mediate retention in the ER and suppress formation of non-native disulfide bonds, such as Bip (Paulsson and Wang, 2003).

3.5. EhPDI has chaperone-like activity One function of chaperones is to suppress aggregation of nonnative forms of proteins during the refolding or unfolding processes (Horwitz et al., 1998). Bona fide chaperones are proteins that promote the complete folding of denatured protein-substrates to its native conformation and are different from chaperone-like proteins which, on the contrary, only retain the ability to prevent

4. Discussion

Fig. 5. pH dependence of the reductase and oxidase activities of recombinant EhPDI. Disulfide reduction of bovine insulin (open circle) or RNAse A oxidative refolding (open square), both assisted by EhPDI, were evaluated at different pH values. Activity assays were carried out as described in materials and methods. Each point represents the mean of three independents (bar, standard error of the mean).

A common feature of some organisms, such as human and yeast, is the expression of a repertoire of PDI enzymes with different domain organization and physiological functions (Kimura et al., 2004; Gruber et al., 2006). However, it has been observed that several substrates did not have specific needs (in terms of folding) and these could be fulfilled by a single and essential PDI, as in yeast (Farquhar et al., 1991; Nørgaard et al., 2001). Interestingly, E. histolytica contains a gene family encoding PDI proteins (Ramos et al., 2008). From a set of 11 polypeptide sequences, six of them display the characteristic features of functional enzymes. EhPDI, a 38-KDa protein from the amebic PDI family, is likely to be the major PDI enzyme involved in protein folding in E. histolytica: (i) it displays the structural features observed in other functional homologues, such as the Dictyostelium discoideum PDI (Monnat et al., 1997), (ii) it is actively expressed in the trophozoite stage, and (iii) it exhibits in vivo oxidase activity (Ramos et al., 2005). Moreover, data from two highly informative transcriptomic analyses showed that the expression levels of EhPDI remain unchanged during intestinal colonization/invasion (Gilchrist et al., 2006) and it is expressed in both stages, trophozoite and cyst, but being not specific to any (Ehrenkaufer et al., 2007), suggesting that EhPDI is important for some cellular functions of the E. histolytica lifestyle.

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EhPDI was also identified as part of a unique set of early phagosomal proteins (Boettner et al., 2008). This observation is intriguing, since the major function of PDI enzymes is related to the ER and is likely to be found in compartments of the secretory pathway (Yoshimori et al., 1990). However, the presence of ER-related proteins in phagosomes has been reported in other cell types, such as neutrophils and macrophages. Apparently, PDI and other ER-resident proteins are important for the formation and maturation of phagosomes (Burlak et al., 2006; Santos et al., 2009). Although the role of EhPDI in amebic phagosomes remains to be determined, it seems that E. histolytica requires the participation of different sets of proteins to accomplish one of its main pathogenic features, phagocytosis (Debnath et al., 2007; Boettner et al., 2008). Amebiasis is the second most prevalent protozoan parasite infection in the world (WHO, 1997). Metronidazole is a highly effective amebicide; however, the possibility of developing drug resistance is a prevalent concern (Lossani et al., 2009; Sato et al., 2010). Moreover, it is advisable following metronidazole treatment with a luminal amebicide to prevent relapse (Haque et al., 2003; Rossignol et al., 2007). Therefore, development of specific and alternative anti-amebic agents is an important issue (Byington et al., 1997; Singh et al., 2008). Several antibiotics have been tested to treat amebiasis (Most et al., 1950; Seneca, 1955; Ghione, 1984). Among them, the peptide antibiotic bacitracin has demonstrated to be an effective drug against E. histolytica (Andrews and Bjorvatn, 1994; Andrews et al., 1995). On the other hand, bacitracin has been extensively used to inhibit PDI enzymes (Roth, 1981; Mizunaga et al., 1990; Mandel et al., 1993). However, the role of this antibiotic as specific inhibitor has been recently disputed. The results from in vitro assays showed that bacitracin has no significant effect on both activities oxidoreductase and chaperone of human PDI (Karala and Ruddock, 2010). In contrast, the inhibition mechanism is apparently through competition for substrate binding (Versteeg and Ruf, 2007; Karala and Ruddock, 2010). In our experimental conditions, bacitracin has shown to be an effective inhibitor of the EhPDI oxidoreductase activities; therefore, is plausible to consider the development of PDI inhibitors as alternative therapy for amebiasis. Although, it remains to be demonstrated whether EhPDI represents a potential drug target, the notion that the inhibition of EhPDI could deprive E. histolytica of functional proteins essential for its survival and parasitic lifestyle is rather attractive. Interestingly, this idea has been supported by inhibition studies on other parasites (Ben Achour et al., 2002; Mouray et al., 2007; Müller et al., 2008). Furthermore, a detailed functional analysis should be performed, such as knock down or silencing experiments, to evaluate the role of this enzyme in the E. histolytica life style, including virulence and survival. Acknowledgments We wish to thank Elisa Noriega and Gabriela Monroy for their excellent technical assistance. This work was supported by a CONACYT Grant (SEP-2004-C01-47554). MAR, REM and SGML are National Researchers (SNI-CONACYT) and members of the Biological–Pharmaceutical Academic Group (Health Sciences, UABC). References Andrews, B.J., Bjorvatn, B., 1994. Chemotherapy of Entamoeba histolytica: studies in vitro with bacitracin and its zinc salt. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 98–100. Andrews, B.J., Nkya, W.M.M.M., Bjorvatn, B., Rønnevig, J.R., 1995. Bacitracin zinc for intestinal amoebiasis: a dose-response study. Current Therapeutic Research 56, 617–625. Ben Achour, Y., Chenik, M., Louzir, H., Dellagi, K., 2002. Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor. Infection and Immunity 70, 3576–3585.

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