# Analysis of the Isomerase and Chaperone-Like Activities of an Amebic PDI (EhPDI)

Authors: Rosa E. Mares; Alexis Z. Minchaca; Salvador Villagrana; Samuel G. Meléndez-López; Marco A. Ramos
Journal: BioMed Research International
 (2015)

---

## Abstract

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. TheEhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation in E. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant of E. coli, to test whether EhPDI exhibits isomerase activity in vivo. Our preliminary results showed that EhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed that EhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization of EhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding in E. histolytica.


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## Body

## 1. Introduction

In eukaryotic cells, folding and posttranslational modifications of proteins are the primary function of the endoplasmic reticulum (ER) [1]. Formation of disulfide bonds, a common modification observed in several secretory proteins, takes place mainly in that compartment [1, 2]. Almost all organisms have a set of proteins involved in folding; however, the cellular and molecular details of this process have been elucidated only in a few model systems, such as the yeast ER and the bacterial periplasmic compartment [3, 4].Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins [5]. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. Some organisms, such as yeast and mammals, have a family of PDI homologues that exhibit distinct domain organization and function [6–8].Under physiological conditions, the cellular mechanisms that respond to proteotoxic stress remain in an inactive state; however, under stressful conditions, several response mechanisms are triggered to restore proteome stability, but if these fail, the apoptotic pathways are activated, leading ultimately to cell death [9, 10]. In addition to assisting oxidative folding of nascent polypeptides, PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins under stressful conditions [10]. Furthermore, since the blocking of PDI activity could lead to protein misfolding, prolonged proteotoxic stress and apoptosis [11, 12] considering PDI as a therapeutic target to stop the progression of some diseases seem plausible [13, 14].Human amebiasis, the parasitic infection caused by the protozoanEntamoeba histolytica, is a prevalent infection in developing countries [15]. Interestingly, the continued secretion of proteins, including virulence factors, is well recognized as the primary feature of this parasite [16]. Moreover, protein folding and correct disulfide bond formation are essential for secreted virulence factors, such as the Gal/GalNAc-inhibitable lectin [17] and the pore-forming peptide A (amoebapore A) [18].TheE. histolytica genome has 11 genes encoding PDI homologues [19]. From these, the enzyme namedEhPDI (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to study protein folding and disulfide bond formation inE. histolytica. By usingin vitro assays and standard substrates (such as insulin, lysozyme, and ribonuclease A), we have confirmed thatEhPDI exhibits the distinctive oxidoreductase activities (reductase, oxidase, and isomerase) as well as the typical chaperone-like function (suppression of polypeptide aggregation) [20, 21]. Only the oxidase activity has been demonstratedin vivo, through functional complementation of thedsbA mutation inE. coli [22].Here, to test whetherEhPDI exhibits isomerase activityin vivo, we performed a functional complementation assay using the ΔdsbC mutant ofE. coli as a model and the defective expression of the periplasmic protein AppA as the phenotype. The acid phosphatase-phytase enzyme (AppA) has three consecutive disulfide bonds and one nonconsecutive that renders it dependent on DsbC [23]. Our preliminary results showed thatEhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Finally, additional studies confirmed thatEhPDI protects two heat-labile enzymes, α-glucosidase andNdeI endonuclease, against thermal inactivation, extending our knowledge about its chaperone-like activity.


## 2. Materials and Methods

### 2.1. Materials

DNA amplification reagents and DNA purification kits were from Qiagen (Valencia, CA). Bacterial media were from Becton Dickinson (Franklin Lakes, NJ). Electrophoresis reagents were from Bio-Rad (Hercules, CA). Endonucleases and other enzymes were from New England Biolabs (Ipswich, MA). Other biochemicals were from Sigma-Aldrich (St. Louis, MO), otherwise mentioned in the text. All reagents used were analytical or molecular biology grade.


### 2.2. Bacterial Strains, Plasmids, and Growth Conditions

Escherichia coli strains and plasmids used in this study are listed in Table 1. Bacterial cultures were grown in LB medium at 37°C, with appropriate antibiotics (ampicillin at 150 μg/mL and chloramphenicol at 15 μg/mL). Recombinant plasmids were constructed by using standard molecular cloning protocols.Table 1
Strains and plasmids used in this study.
Strains or plasmids
Relevant genotype or features
Source or reference
Strains
XL1-Blue MRF′
Δ
(
mcrA
)
183 Δ
(
mcrCB-hsdSMR-mrr
)
173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac
[
F
′
proAB
l
a
c
I
q
ZΔM15 Tn10
(
T
e
t
R
)
]
Stratagene
Shuffle Express
fhuA2
[
lon
]
ompT ahpC galλatt::pNEB3-r1-cDsbC
(
S
p
e
c
R
,
l
a
c
I
q
)
ΔtrxB sulA11 R
(
mcr-73::miniTn10–
T
e
t
S
)
2
[
dcm
]
R
(
zgb-210::Tn10–
T
e
t
S
)
endA1 Δgor Δ
(
mcrC-mrr
)
114::IS10
NEB1
BW25113
F
-
Δ
(
araD-araB
)
567  ΔlacZ4787(::rrnB-3) λ-rph-1  Δ(rhaD-rhaB)568  hsdR514
CGSC2  [54]
JW2861-1
BW25113dsbC744::kan
GCSC2  [54]
Plasmid
pBAD33
Arabinose regulation, p15A origin,CmR
ATCC3  [55]
pBAD-AppA
pBAD33-based, periplasmic AppA
This study
pBluescript SK-
Lactose regulation, ColE1 origin,AmpR
Stratagene
pBAppA
pBluescript-based, periplasmic AppA
This study
pBPelB-EhPDI
pBluescript-based, periplasmicEhPDI (wild type)
[22]
pBRM05
pBluescript-based, periplasmicEhPDISS/CC (C44S; C47S)
[20]
pBRM06
pBluescript-based, periplasmicEhPDICC/SS (C160S; C163S)
[20]
pBRM15
pBluescript-based, periplasmicEhPDISS/SS (C44S; C47S; C160S; C163S)
[20]
pQE30
Lactose regulation, ColE1 origin,AmpR
Qiagen
pQHPDI
pQE30-based, recombinantEhPDI (wild type)
[20]
pQRM05
pQE30-based, recombinantEhPDISS/CC (C44S; C47S)
This study
pQRM06
pQE30-based, recombinantEhPDICC/SS (C160S; C163S)
This study
pQRM15
pQE30-based, recombinantEhPDISS/SS (C44S; C47S; C160S; C163S)
This study
1New England Biolabs; 2Coli Genetic Stock Center; 3American Type Culture Collection.


#### 2.2.1. Construction of pBAD-AppA Plasmid

Full-length sequence of the bacterialappA gene was amplified from genomic DNA (XL1-Blue MRF′ strain), using the synthetic oligonucleotides EcAppAF (5′-cgc gcg gaa ttc ATG AAA AGC GGA AAC ATA TCG-3′) and EcAppAR (5′-cgc gcg tct aga TTA CAA ACT GCA CGC CGG TAT-3′) as primers. The PCR product was then digested withEcoRI andXbaI endonucleases and cloned into theEcoRI-XbaI sites of pBluescript SK-, yielding the pBAppA plasmid. To obtain the pBAD-AppA plasmid, a site forEcoRV (located immediately upstream of theEcoRI) was used to get a restriction fragment fromEcoRV-XbaI sites, which was then subcloned intoSmaI-XbaI sites of pBAD33. TheappA gene was confirmed by DNA sequencing.


#### 2.2.2. Construction of the pQRM05, pQRM06, and pQRM15 Plasmids

TheEhPDI gene variants (with Cys to Ser substitutions) were amplified from its corresponding pBluescript-based plasmid (Table 1), using the synthetic oligonucleotidesEhPDIp38F (5′-cat cac gga tcc GCT GAT GTA GTA TCA TTA AAT C-3′) and M13FW (5′-GTA AAA CGA CGG CCA GTG-3′) as primers. Then, PCR products were digested withBamHI andHindIII endonucleases and subcloned into the same sites of pQE30 (in frame with the sequence encoding the N-terminal hexahistidine tag). TheEhPDI gene variants were confirmed by DNA sequencing.


### 2.3. DsbC Complementation and AppA Activity Assay

#### 2.3.1. Periplasmic Expression of AppA and Coexpression withEhPDI

E. coli strains BW25113 (wild type) and JW2861-1 (ΔdsbC mutant) were transformed with pBAD33 (as control) or pBAD-AppA. Stable transformants were cultured in LB medium, supplemented with chloramphenicol, and the periplasmic expression of AppA was induced with 0.2% arabinose. Bacterial cell pellets (from 1 mL) were obtained by centrifugation (2 min at 10,000 rpm).E. coli strain JW2861-1 harboring pBAD-AppA was transformed with pBluescript-based plasmids expressingEhPDI variants (Table 1). The plasmid pBluescript SK- was used as a control. Stable cotransformants were cultured in LB medium, supplemented with ampicillin and chloramphenicol, and the periplasmic coexpression of AppA andEhPDI was induced with 0.2% arabinose and 1 mM IPTG. Bacterial cell pellets were obtained as before.


#### 2.3.2. Acid Phosphatase Activity Assay

The acid phosphatase activity was determined by a colorimetric assay [23]. Bacterial cell pellets were resuspended in glycine buffer (0.25 M; pH 2.5) and adjusted to 0.3–0.6 units ofA600 per mL. Then, 20 μL aliquots were further diluted with 80 μL of the same buffer and mixed with 100 μL of 50 mMp-nitrophenyl phosphate. After 15 min of incubation at 37°C, reactions were stopped by adding 1 mL of 1.2 N NaOH. Immediately, supernatants were separated by centrifugation (5 min at 14,500 rpm) and the releasedp-nitrophenolate was quantified by measuring theA420. Light scattering by cellular debris was also considered (recording theA550). The acid phosphatase activity was expressed in Miller units [23, 24].


### 2.4. Purification of RecombinantEhPDI Enzymes

E. coli strain Shuffle Express was transformed with pQE30-based plasmids expressing recombinantEhPDI enzyme variants (see Table 1). Stable transformants were cultured in LB medium, supplemented with ampicillin, and protein expression was induced with 0.1 mM IPTG. Bacterial cells (from 100 mL) were harvested and lysed under native conditions, using the CelLytic B Plus Kit (Sigma-Aldrich). From the soluble fraction, recombinant proteins were purified by Ni-affinity chromatography (The QIAexpressionist, Qiagen). Eluate fractions were analyzed by SDS-PAGE and those containing more than 95% of pure protein were pooled and concentrated/desalted by ultrafiltration, using a Microsep UF Spin Filter (Pall Co.). Protein concentration was determined by performing the BCA colorimetric assay (Sigma-Aldrich), using BSA as standard.


### 2.5. Oxidative Refolding Assay

Oxidative refolding of denatured-reduced ribonuclease A (drRNAse) by recombinant amebic PDI enzymes was assayed by following a reported protocol [25]. Refolding was achieved by diluting drRNAse (7.8 μM) into a reaction buffer (2 mM GSH, 0.4 mM GSSG, 100 mm Tris-HCl, pH 8.0) containing 5 μM of amebic PDI enzymes and 4.5 mM of cCMP (RNAse substrate). The reactivation of RNAse was followed for 60 min by recording the absorbance at 296 nm. Active RNAse (μM) was calculated from the first derivative of the absorbance over time and corrected for the depletion of the substrate and the formation of the product (CMP, RNAse inhibitor) [25]. The isomerase activity was determined from the linear increase of active RNAse over time (μM/min), after the lag phase (which reflects the oxidase activity, min−1).


### 2.6. Disulfide Reductase Assay

Disulfide reduction of bovine insulin catalyzed by recombinant amebic PDI enzymes was assayed according to a standard turbidity method [21, 26]. Recombinant enzymes (2 μM final) were added to a reaction buffer (2 mM EDTA and 100 mM HEPES; pH 7.0) containing bovine insulin (100 μM final). Disulfide reduction was started by adding DTT (0.3 mM final) and followed for 90 min by recording theA650 every 5 min. Reductase activity was determined from the linear increase of absorbance over time after the lag phase (A650/min2) [27].


### 2.7. Chaperone Activity Assays

Chaperone-like activity ofEhPDI was evaluated by performing a protection against thermal inactivation assay of two heat-labile enzymes: α-glucosidase [28, 29] andNdeI endonuclease [30, 31].


#### 2.7.1. Thermal Inactivation ofα-Glucosidase

Different concentrations ofEhPDI (0–5 μM final) were added to a reaction buffer (50 mM KH2PO4; pH 6.8) containing yeast α-glucosidase (16 μg/mL final). Thermal inactivation was performed by incubating at 43°C for 60 min (a control without treatment was carried out for each concentration). Then, reactions were cooled on ice for 1 min and the aggregated protein was separated by centrifugation (5 min at 14,500 rpm). α-glucosidase activity was determined by diluting a 40 μL aliquot of the supernatant with 160 μL of reaction buffer containing 0.125 mM of reduced glutathione and 1.25 mM ofp-nitrophenyl-α-D-glucopyranoside. After 20 min of incubation at room temperature, reactions were stopped by adding 50 μL of 0.5 M Na2CO3. The releasedp-nitrophenolate was quantified by measuring the absorbance at 415 nm. The α-glucosidase activity (AG) was defined by the increase in absorbance over time (A415/min). For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=(AGT/AGU)×100, where AGT represents the remaining α-glucosidase activity after thermal treatment (60 min at 43°C), while the untreated enzyme is represented by AGU.


#### 2.7.2. Thermal Inactivation ofNdeI Endonuclease

Different concentrations ofEhPDI (0–2 μM final) were added to a reaction buffer (75 mM potassium acetate, 30 mM Tris-acetate, 15 mM magnesium acetate, 1.5 mM DTT; pH 7.9) containingNdeI endonuclease (1 U/μL final). Thermal inactivation was performed by incubating at 50°C for 30 min (control reactions without incubation were carried out). Then, the reactions were cooled on ice for 2 min and briefly centrifuged (to collect a 10 μL volume). Endonucleolytic activity was determined by adding 5 μL of a plasmid solution, containing 0.1 μg of pUC19 and 0.1 mg of BSA. After 2 hours of incubation at 37°C, standard agarose gel electrophoresis and ethidium bromide staining were used to analyze the restriction fragments. TheNdeI endonuclease activity was defined by the relative amount of linearized plasmid, estimated by digital densitometry. For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=[(ENT-C2)/(C1-C2)]×100, where ENT represents the remainingNdeI endonuclease activity after thermal treatment (30 min at 50°C), while C1 (withNdeI) and C2 (withoutNdeI) correspond to the control reactions without treatment.


### 2.8. Statistical Analysis

Unless otherwise mentioned, activity data were from three independent experiments and are represented as mean ± standard error. All statistical analysis were performed using Prism v.5 (GraphPad Software, San Diego, CA). Unpairedt-test was used for routine comparison of data sets. P values less than 0.05 were considered statistically significant.


## 2.1. Materials

DNA amplification reagents and DNA purification kits were from Qiagen (Valencia, CA). Bacterial media were from Becton Dickinson (Franklin Lakes, NJ). Electrophoresis reagents were from Bio-Rad (Hercules, CA). Endonucleases and other enzymes were from New England Biolabs (Ipswich, MA). Other biochemicals were from Sigma-Aldrich (St. Louis, MO), otherwise mentioned in the text. All reagents used were analytical or molecular biology grade.


## 2.2. Bacterial Strains, Plasmids, and Growth Conditions

Escherichia coli strains and plasmids used in this study are listed in Table 1. Bacterial cultures were grown in LB medium at 37°C, with appropriate antibiotics (ampicillin at 150 μg/mL and chloramphenicol at 15 μg/mL). Recombinant plasmids were constructed by using standard molecular cloning protocols.Table 1
Strains and plasmids used in this study.
Strains or plasmids
Relevant genotype or features
Source or reference
Strains
XL1-Blue MRF′
Δ
(
mcrA
)
183 Δ
(
mcrCB-hsdSMR-mrr
)
173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac
[
F
′
proAB
l
a
c
I
q
ZΔM15 Tn10
(
T
e
t
R
)
]
Stratagene
Shuffle Express
fhuA2
[
lon
]
ompT ahpC galλatt::pNEB3-r1-cDsbC
(
S
p
e
c
R
,
l
a
c
I
q
)
ΔtrxB sulA11 R
(
mcr-73::miniTn10–
T
e
t
S
)
2
[
dcm
]
R
(
zgb-210::Tn10–
T
e
t
S
)
endA1 Δgor Δ
(
mcrC-mrr
)
114::IS10
NEB1
BW25113
F
-
Δ
(
araD-araB
)
567  ΔlacZ4787(::rrnB-3) λ-rph-1  Δ(rhaD-rhaB)568  hsdR514
CGSC2  [54]
JW2861-1
BW25113dsbC744::kan
GCSC2  [54]
Plasmid
pBAD33
Arabinose regulation, p15A origin,CmR
ATCC3  [55]
pBAD-AppA
pBAD33-based, periplasmic AppA
This study
pBluescript SK-
Lactose regulation, ColE1 origin,AmpR
Stratagene
pBAppA
pBluescript-based, periplasmic AppA
This study
pBPelB-EhPDI
pBluescript-based, periplasmicEhPDI (wild type)
[22]
pBRM05
pBluescript-based, periplasmicEhPDISS/CC (C44S; C47S)
[20]
pBRM06
pBluescript-based, periplasmicEhPDICC/SS (C160S; C163S)
[20]
pBRM15
pBluescript-based, periplasmicEhPDISS/SS (C44S; C47S; C160S; C163S)
[20]
pQE30
Lactose regulation, ColE1 origin,AmpR
Qiagen
pQHPDI
pQE30-based, recombinantEhPDI (wild type)
[20]
pQRM05
pQE30-based, recombinantEhPDISS/CC (C44S; C47S)
This study
pQRM06
pQE30-based, recombinantEhPDICC/SS (C160S; C163S)
This study
pQRM15
pQE30-based, recombinantEhPDISS/SS (C44S; C47S; C160S; C163S)
This study
1New England Biolabs; 2Coli Genetic Stock Center; 3American Type Culture Collection.


### 2.2.1. Construction of pBAD-AppA Plasmid

Full-length sequence of the bacterialappA gene was amplified from genomic DNA (XL1-Blue MRF′ strain), using the synthetic oligonucleotides EcAppAF (5′-cgc gcg gaa ttc ATG AAA AGC GGA AAC ATA TCG-3′) and EcAppAR (5′-cgc gcg tct aga TTA CAA ACT GCA CGC CGG TAT-3′) as primers. The PCR product was then digested withEcoRI andXbaI endonucleases and cloned into theEcoRI-XbaI sites of pBluescript SK-, yielding the pBAppA plasmid. To obtain the pBAD-AppA plasmid, a site forEcoRV (located immediately upstream of theEcoRI) was used to get a restriction fragment fromEcoRV-XbaI sites, which was then subcloned intoSmaI-XbaI sites of pBAD33. TheappA gene was confirmed by DNA sequencing.


### 2.2.2. Construction of the pQRM05, pQRM06, and pQRM15 Plasmids

TheEhPDI gene variants (with Cys to Ser substitutions) were amplified from its corresponding pBluescript-based plasmid (Table 1), using the synthetic oligonucleotidesEhPDIp38F (5′-cat cac gga tcc GCT GAT GTA GTA TCA TTA AAT C-3′) and M13FW (5′-GTA AAA CGA CGG CCA GTG-3′) as primers. Then, PCR products were digested withBamHI andHindIII endonucleases and subcloned into the same sites of pQE30 (in frame with the sequence encoding the N-terminal hexahistidine tag). TheEhPDI gene variants were confirmed by DNA sequencing.


## 2.2.1. Construction of pBAD-AppA Plasmid

Full-length sequence of the bacterialappA gene was amplified from genomic DNA (XL1-Blue MRF′ strain), using the synthetic oligonucleotides EcAppAF (5′-cgc gcg gaa ttc ATG AAA AGC GGA AAC ATA TCG-3′) and EcAppAR (5′-cgc gcg tct aga TTA CAA ACT GCA CGC CGG TAT-3′) as primers. The PCR product was then digested withEcoRI andXbaI endonucleases and cloned into theEcoRI-XbaI sites of pBluescript SK-, yielding the pBAppA plasmid. To obtain the pBAD-AppA plasmid, a site forEcoRV (located immediately upstream of theEcoRI) was used to get a restriction fragment fromEcoRV-XbaI sites, which was then subcloned intoSmaI-XbaI sites of pBAD33. TheappA gene was confirmed by DNA sequencing.


## 2.2.2. Construction of the pQRM05, pQRM06, and pQRM15 Plasmids

TheEhPDI gene variants (with Cys to Ser substitutions) were amplified from its corresponding pBluescript-based plasmid (Table 1), using the synthetic oligonucleotidesEhPDIp38F (5′-cat cac gga tcc GCT GAT GTA GTA TCA TTA AAT C-3′) and M13FW (5′-GTA AAA CGA CGG CCA GTG-3′) as primers. Then, PCR products were digested withBamHI andHindIII endonucleases and subcloned into the same sites of pQE30 (in frame with the sequence encoding the N-terminal hexahistidine tag). TheEhPDI gene variants were confirmed by DNA sequencing.


## 2.3. DsbC Complementation and AppA Activity Assay

### 2.3.1. Periplasmic Expression of AppA and Coexpression withEhPDI

E. coli strains BW25113 (wild type) and JW2861-1 (ΔdsbC mutant) were transformed with pBAD33 (as control) or pBAD-AppA. Stable transformants were cultured in LB medium, supplemented with chloramphenicol, and the periplasmic expression of AppA was induced with 0.2% arabinose. Bacterial cell pellets (from 1 mL) were obtained by centrifugation (2 min at 10,000 rpm).E. coli strain JW2861-1 harboring pBAD-AppA was transformed with pBluescript-based plasmids expressingEhPDI variants (Table 1). The plasmid pBluescript SK- was used as a control. Stable cotransformants were cultured in LB medium, supplemented with ampicillin and chloramphenicol, and the periplasmic coexpression of AppA andEhPDI was induced with 0.2% arabinose and 1 mM IPTG. Bacterial cell pellets were obtained as before.


### 2.3.2. Acid Phosphatase Activity Assay

The acid phosphatase activity was determined by a colorimetric assay [23]. Bacterial cell pellets were resuspended in glycine buffer (0.25 M; pH 2.5) and adjusted to 0.3–0.6 units ofA600 per mL. Then, 20 μL aliquots were further diluted with 80 μL of the same buffer and mixed with 100 μL of 50 mMp-nitrophenyl phosphate. After 15 min of incubation at 37°C, reactions were stopped by adding 1 mL of 1.2 N NaOH. Immediately, supernatants were separated by centrifugation (5 min at 14,500 rpm) and the releasedp-nitrophenolate was quantified by measuring theA420. Light scattering by cellular debris was also considered (recording theA550). The acid phosphatase activity was expressed in Miller units [23, 24].


## 2.3.1. Periplasmic Expression of AppA and Coexpression withEhPDI

E. coli strains BW25113 (wild type) and JW2861-1 (ΔdsbC mutant) were transformed with pBAD33 (as control) or pBAD-AppA. Stable transformants were cultured in LB medium, supplemented with chloramphenicol, and the periplasmic expression of AppA was induced with 0.2% arabinose. Bacterial cell pellets (from 1 mL) were obtained by centrifugation (2 min at 10,000 rpm).E. coli strain JW2861-1 harboring pBAD-AppA was transformed with pBluescript-based plasmids expressingEhPDI variants (Table 1). The plasmid pBluescript SK- was used as a control. Stable cotransformants were cultured in LB medium, supplemented with ampicillin and chloramphenicol, and the periplasmic coexpression of AppA andEhPDI was induced with 0.2% arabinose and 1 mM IPTG. Bacterial cell pellets were obtained as before.


## 2.3.2. Acid Phosphatase Activity Assay

The acid phosphatase activity was determined by a colorimetric assay [23]. Bacterial cell pellets were resuspended in glycine buffer (0.25 M; pH 2.5) and adjusted to 0.3–0.6 units ofA600 per mL. Then, 20 μL aliquots were further diluted with 80 μL of the same buffer and mixed with 100 μL of 50 mMp-nitrophenyl phosphate. After 15 min of incubation at 37°C, reactions were stopped by adding 1 mL of 1.2 N NaOH. Immediately, supernatants were separated by centrifugation (5 min at 14,500 rpm) and the releasedp-nitrophenolate was quantified by measuring theA420. Light scattering by cellular debris was also considered (recording theA550). The acid phosphatase activity was expressed in Miller units [23, 24].


## 2.4. Purification of RecombinantEhPDI Enzymes

E. coli strain Shuffle Express was transformed with pQE30-based plasmids expressing recombinantEhPDI enzyme variants (see Table 1). Stable transformants were cultured in LB medium, supplemented with ampicillin, and protein expression was induced with 0.1 mM IPTG. Bacterial cells (from 100 mL) were harvested and lysed under native conditions, using the CelLytic B Plus Kit (Sigma-Aldrich). From the soluble fraction, recombinant proteins were purified by Ni-affinity chromatography (The QIAexpressionist, Qiagen). Eluate fractions were analyzed by SDS-PAGE and those containing more than 95% of pure protein were pooled and concentrated/desalted by ultrafiltration, using a Microsep UF Spin Filter (Pall Co.). Protein concentration was determined by performing the BCA colorimetric assay (Sigma-Aldrich), using BSA as standard.


## 2.5. Oxidative Refolding Assay

Oxidative refolding of denatured-reduced ribonuclease A (drRNAse) by recombinant amebic PDI enzymes was assayed by following a reported protocol [25]. Refolding was achieved by diluting drRNAse (7.8 μM) into a reaction buffer (2 mM GSH, 0.4 mM GSSG, 100 mm Tris-HCl, pH 8.0) containing 5 μM of amebic PDI enzymes and 4.5 mM of cCMP (RNAse substrate). The reactivation of RNAse was followed for 60 min by recording the absorbance at 296 nm. Active RNAse (μM) was calculated from the first derivative of the absorbance over time and corrected for the depletion of the substrate and the formation of the product (CMP, RNAse inhibitor) [25]. The isomerase activity was determined from the linear increase of active RNAse over time (μM/min), after the lag phase (which reflects the oxidase activity, min−1).


## 2.6. Disulfide Reductase Assay

Disulfide reduction of bovine insulin catalyzed by recombinant amebic PDI enzymes was assayed according to a standard turbidity method [21, 26]. Recombinant enzymes (2 μM final) were added to a reaction buffer (2 mM EDTA and 100 mM HEPES; pH 7.0) containing bovine insulin (100 μM final). Disulfide reduction was started by adding DTT (0.3 mM final) and followed for 90 min by recording theA650 every 5 min. Reductase activity was determined from the linear increase of absorbance over time after the lag phase (A650/min2) [27].


## 2.7. Chaperone Activity Assays

Chaperone-like activity ofEhPDI was evaluated by performing a protection against thermal inactivation assay of two heat-labile enzymes: α-glucosidase [28, 29] andNdeI endonuclease [30, 31].


### 2.7.1. Thermal Inactivation ofα-Glucosidase

Different concentrations ofEhPDI (0–5 μM final) were added to a reaction buffer (50 mM KH2PO4; pH 6.8) containing yeast α-glucosidase (16 μg/mL final). Thermal inactivation was performed by incubating at 43°C for 60 min (a control without treatment was carried out for each concentration). Then, reactions were cooled on ice for 1 min and the aggregated protein was separated by centrifugation (5 min at 14,500 rpm). α-glucosidase activity was determined by diluting a 40 μL aliquot of the supernatant with 160 μL of reaction buffer containing 0.125 mM of reduced glutathione and 1.25 mM ofp-nitrophenyl-α-D-glucopyranoside. After 20 min of incubation at room temperature, reactions were stopped by adding 50 μL of 0.5 M Na2CO3. The releasedp-nitrophenolate was quantified by measuring the absorbance at 415 nm. The α-glucosidase activity (AG) was defined by the increase in absorbance over time (A415/min). For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=(AGT/AGU)×100, where AGT represents the remaining α-glucosidase activity after thermal treatment (60 min at 43°C), while the untreated enzyme is represented by AGU.


### 2.7.2. Thermal Inactivation ofNdeI Endonuclease

Different concentrations ofEhPDI (0–2 μM final) were added to a reaction buffer (75 mM potassium acetate, 30 mM Tris-acetate, 15 mM magnesium acetate, 1.5 mM DTT; pH 7.9) containingNdeI endonuclease (1 U/μL final). Thermal inactivation was performed by incubating at 50°C for 30 min (control reactions without incubation were carried out). Then, the reactions were cooled on ice for 2 min and briefly centrifuged (to collect a 10 μL volume). Endonucleolytic activity was determined by adding 5 μL of a plasmid solution, containing 0.1 μg of pUC19 and 0.1 mg of BSA. After 2 hours of incubation at 37°C, standard agarose gel electrophoresis and ethidium bromide staining were used to analyze the restriction fragments. TheNdeI endonuclease activity was defined by the relative amount of linearized plasmid, estimated by digital densitometry. For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=[(ENT-C2)/(C1-C2)]×100, where ENT represents the remainingNdeI endonuclease activity after thermal treatment (30 min at 50°C), while C1 (withNdeI) and C2 (withoutNdeI) correspond to the control reactions without treatment.


## 2.7.1. Thermal Inactivation ofα-Glucosidase

Different concentrations ofEhPDI (0–5 μM final) were added to a reaction buffer (50 mM KH2PO4; pH 6.8) containing yeast α-glucosidase (16 μg/mL final). Thermal inactivation was performed by incubating at 43°C for 60 min (a control without treatment was carried out for each concentration). Then, reactions were cooled on ice for 1 min and the aggregated protein was separated by centrifugation (5 min at 14,500 rpm). α-glucosidase activity was determined by diluting a 40 μL aliquot of the supernatant with 160 μL of reaction buffer containing 0.125 mM of reduced glutathione and 1.25 mM ofp-nitrophenyl-α-D-glucopyranoside. After 20 min of incubation at room temperature, reactions were stopped by adding 50 μL of 0.5 M Na2CO3. The releasedp-nitrophenolate was quantified by measuring the absorbance at 415 nm. The α-glucosidase activity (AG) was defined by the increase in absorbance over time (A415/min). For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=(AGT/AGU)×100, where AGT represents the remaining α-glucosidase activity after thermal treatment (60 min at 43°C), while the untreated enzyme is represented by AGU.


## 2.7.2. Thermal Inactivation ofNdeI Endonuclease

Different concentrations ofEhPDI (0–2 μM final) were added to a reaction buffer (75 mM potassium acetate, 30 mM Tris-acetate, 15 mM magnesium acetate, 1.5 mM DTT; pH 7.9) containingNdeI endonuclease (1 U/μL final). Thermal inactivation was performed by incubating at 50°C for 30 min (control reactions without incubation were carried out). Then, the reactions were cooled on ice for 2 min and briefly centrifuged (to collect a 10 μL volume). Endonucleolytic activity was determined by adding 5 μL of a plasmid solution, containing 0.1 μg of pUC19 and 0.1 mg of BSA. After 2 hours of incubation at 37°C, standard agarose gel electrophoresis and ethidium bromide staining were used to analyze the restriction fragments. TheNdeI endonuclease activity was defined by the relative amount of linearized plasmid, estimated by digital densitometry. For each concentration ofEhPDI, the percentage of protection against thermal inactivation (chaperone-like activity) was determined by using the following equation: protection (%)=[(ENT-C2)/(C1-C2)]×100, where ENT represents the remainingNdeI endonuclease activity after thermal treatment (30 min at 50°C), while C1 (withNdeI) and C2 (withoutNdeI) correspond to the control reactions without treatment.


## 2.8. Statistical Analysis

Unless otherwise mentioned, activity data were from three independent experiments and are represented as mean ± standard error. All statistical analysis were performed using Prism v.5 (GraphPad Software, San Diego, CA). Unpairedt-test was used for routine comparison of data sets. P values less than 0.05 were considered statistically significant.


## 3. Results and Discussion

### 3.1.EhPDI ExhibitsIn Vivo Isomerase Activity

To study the functional activities of eukaryotic PDI enzymesin vivo, yeast and bacterial cells have been successfully used to complement phenotypes associated with defective formation of disulfide bonds [32, 33]. InE. coli cells, the oxidative folding of polypeptides is carried out in the periplasmic compartment and performed by the Dsb proteins: oxidation and isomerization of disulfide bonds are catalyzed by DsbA and DsbC, respectively [34].The DsbC protein is particularly notable since it shares structural and functional similarities with eukaryotic PDI enzymes [35]. In fact, its functional role as disulfide isomerase has been studied using eukaryotic multidisulfide proteins as substrates [35, 36]. Four physiological substrates of DsbC have been identified so far: AppA [23], RcsF [37], MepA, and RNAse I [38]; from these,in vivo studies using AppA as substrate protein showed that DsbC plays an important role during folding of proteins with nonconsecutive disulfide bonds [23]. Then, to test whetherEhPDI exhibits disulfide isomerase activityin vivo, we performed a functional complementation assay using the ΔdsbC mutant ofE. coli as a model and the defective periplasmic expression of AppA as the phenotype.Initially, the absence of acid phosphatase activity (as background) was confirmed, indicating that the chromosomalappA gene was not induced under our experimental conditions (data not shown). Then, pBAD-AppA (Table 1) was used to transform both wild type and ΔdsbC mutant strains. Stable transformants were cultured and AppA expression was induced properly. As noted in Figure 1, a high level of acid phosphatase activity was detected in the wild type strain, 9595±593 Miller units, whereas a low level of activity was observed in the ΔdsbC mutant (1593±70 Miller units), confirming the DsbC-dependence of AppA (Figure 1). Then, pBPelB-EhPDI (Table 1) was used to transform the ΔdsbC/pBAD-AppA strain, using a two-plasmid system [39]. Stable cotransformants were cultured and protein expression was induced accordingly. Periplasmic expression ofEhPDI was confirmed by immunoblot (data not shown). Interestingly, a significant increase in acid phosphatase activity was detected (5151±344 Miller units), suggesting that the correct disulfide bond formation of AppA was assisted by the isomerase activity ofEhPDI.Figure 1
Acid phosphatase activity when AppA was expressed in the wild type and ΔdsbC mutant strains, as well as when it was coexpressed withEhPDI in the ΔdsbC mutant strain. The activity (expressed in Miller units) is shown on the left. The plasmids used for transfection ofE. coli are also indicated.


### 3.2. Isomerase Activity ofEhPDI Is Limited and Dependent on Its Reductase Activity

The active site of the thioredoxin-like domains from DsbA, DsbC, and PDI enzymes is characterized by the presence of the motif CXXC, where the cysteine residues play an important role in the enzymatic activity [40, 41].EhPDI contains two thioredoxin-like domains (referred to as N- and C-Trx, resp.; both having the motif CGHC) that are essential for itsin vivo oxidase activity [20]. To test whether both domains contribute to the isomerase activity, we carried out mutagenic analysis followed by a functional complementation assay.The pBluescript-based plasmids expressingEhPDI variants (Table 1) were used to transform the ΔdsbC/pBAD-AppA strain. Stable cotransformants were cultured and protein expression was induced accordingly. Periplasmic expression ofEhPDI variants was confirmed by immunoblot (data not shown). As indicated in Table 2, the isomerase activity ofEhPDI is dependent on its CGHC active sites, since a complete loss of the AppA activity was observed when the variant having both domains inactivated was coexpressed (EhPDISS/SS). Also, low AppA activity was detected when each domain was tested without the background of the other (EhPDISS/CC and EhPDICC/SS); furthermore, the slight difference observed between these two variants can be explained by considering that the thioredoxin-like domains are not equivalent with regard to the isomerase activity [33, 42]. However, it is important to take into account the cellular features of the bacterial model to better understand the role ofEhPDI as an isomerasein vivo.Table 2
In vivo isomerase activity of EhPDI enzyme variants.
Enzyme variant
Acid phosphatase (AppA)∗ activity in Miller units (%)¶
EhPDISS/SS
732 ± 74 (−1)
EhPDISS/CC
1086 ± 136 (7)
EhPDICC/SS
1646 ± 230 (20)
∗The data was expressed as ± standard error (n = 6).
¶Under the background of ΔdsbC/pBAD-AppA, normalization was performed considering the mean activity of pBPelB-EhPDI as maximal (Amax⁡ = 5151) and pBluescript SK- as minimal (Amin⁡ = 780). The percentage (%) was calculated as [(A-Amin⁡)/(Amax⁡-Amin⁡)] × 100, where A represents the activity of the enzyme variant.In the periplasmic compartment, substrate proteins with misoxidized disulfide bonds are shuffled to properly oxidized states by two mechanisms: (1) the isomerase pathway, where DsbC acts on the substrate as reductase-oxidase, and (2) the reductase pathway, where DsbC simply acts as reductase, allowing DsbA another chance to correctly oxidize the substrate [43]. In addition, when the ΔdsbC mutant ofE. coli was complemented with the protein TrxP fromBacteroides fragilis (a periplasmic reductase with poor isomerase activity), a fully restored AppA activity was observed, indicating that the disulfide bond isomerization of this substrate is accomplished mainly through the reductase pathway [43]. Hence, it is reasonable to think that the low AppA activity detected when the ΔdsbC mutant ofE. coli was complemented with any of the variants (Table 2) suggests that the isomerase function ofEhPDI is dependent on its reductase activity. To test this, we performed twoin vitro activity assays, oxidative refolding of ribonuclease and reduction of insulin, using purified recombinant enzymes, that is,EhPDI variants.As indicated in Table3, the wild type (EhPDI) and variants (EhPDISS/CC and EhPDICC/SS) showed comparable oxidative refolding capabilities. In contrast, significant differences were observed in their reductive activities: EhPDICC/SS retained about 60%, whilst EhPDISS/CC roughly retained 20%. These results confirmed thatEhPDI is dependent on its reductase activity to function as oxidoreductasein vivo andin vitro.Table 3
In vitro activities of purified EhPDI enzyme variants.
Enzyme
RNAse A oxidative refolding∗†¶
Insulin reduction∗†¶   [×10−6 A650/min2]  (%)
Oxidase[×10−6 min−1] (%)
Isomerase[×10−6 μM/min] (%)
EhPDI
513 ± 16 (100)
39 ± 1 (100)
109 ± 11 (100)
EhPDISS/CC
551 ± 16 (107)
33 ± 1 (85)
22 ± 2 (20)
EhPDICC/SS
517 ± 13 (101)
31 ± 1 (79)
65 ± 5 (60)
∗The data was expressed as mean ± standard error (n = 3).
†The activity of the enzyme variant EhPDISS/SS was not statistically significant, as compared with the reaction performed in the absence of enzyme.
¶The activity ratio (%) was calculated as (variant/wild type) × 100.


### 3.3.EhPDI Protects Proteins against Thermal-Induced Aggregation

The chaperone-like function of PDI enzymes is determined by their ability to protect misfolded/unfolded substrate proteins against thermal-induced aggregation and to assist refolding [44, 45]. This function is essential in order for PDI to act as an efficient folding catalyst, since it allows access to buried thiols and disulfide bonds in the substrates and prevents nonspecific interactions between partially folded intermediates [1]. Typically, the chaperone-like function has been studiedin vitro by measuring the ability to prevent protein aggregation induced by different physical or chemical conditions, such as temperature or denaturants [5, 46].We have already reported thatEhPDI exhibits chaperone-like function by showing its ability to prevent the DTT-induced aggregation of the B chain of insulin [21]. Although this assay was a simple approach to test the chaperone-like function ofEhPDI, the low molecular mass of the substrate (3.4 kDa) represents a limitation of this assay, since it offers a restricted number of contact sites to form stable complexes [47]. Hence, to gain further insights regarding the chaperone-like function ofEhPDI, we performed additionalin vitro assays to test its ability to prevent thermal-induced aggregation, using as substrates two heat-labile enzymes: α-glucosidase andNdeI endonuclease.As shown in Figures2 and 3, the chaperone-like function ofEhPDI was dose-dependent, since an increment of activity was observed as a result of augmenting its concentration. Moreover, to estimate its chaperone-like ability, the half-maximal effective concentration (EC50) and Hill slope were calculated by fitting the data to a model of one specific binding site with a variable slope. Interestingly, the apparent values obtained for α-glucosidase (EC50 = 3.0±0.4 μM; Hill slope = 1.0) andNdeI endonuclease (EC50 = 0.26±0.06 μM; Hill slope = 2.4) suggest thatEhPDI exhibits differences in substrate specificity and affinity [48].Figure 2
Protection of thermal inactivation ofα-glucosidase assisted byEhPDI. Relative chaperone-like activity (%) of recombinantEhPDI.Figure 3
Protection of thermal inactivation ofNdeI endonuclease assisted byEhPDI. Relative chaperone-like activity (%) of recombinantEhPDI. Upper panel: agarose gel indicating the relative mobility of the linearized and supercoiled plasmid (C1 and C2); also, concentrations ofEhPDI are indicated.AlthoughEhPDI does not have a substrate-binding b′-domain (a-a′-D) as the mammalian homologue (a-b-b′-a′-c) [49], the notion that some other domains (e.g., a′ or D) might be involved in the chaperone activity is evident. This idea is supported by the results of two previous reports: (i) ERp46, which lacks a b′-domain, is able to bind peptides through its catalytic domains (a0, a, and a′) [50] and (ii) the D-domain of ERp29 contains a discrete and conserved substrate-binding site [51].


### 3.4. Closing Remarks

Although little is known about theE. histolytica mechanisms that act in response to proteotoxic stress [52], the upregulation of genes encoding typical chaperones (such as Hsp70 and Hsp90) in a response to thermal stress suggests that it contains the cellular machinery necessary to preserve and restore the stability of the proteome [53]. So, the identification and characterization ofEhPDI as a folding catalyst with chaperone-like activity represents an additional step to dissect the molecular mechanisms involved in both protein folding and proteotoxic stress inE. histolytica. Hence, it is conceivable to suppose that inhibition ofEhPDI could lead to an increase in protein misfolding, promoting a sustained proteotoxic stress, eventually inducing apoptosis and, thus, preventing infection by this parasite.


## 3.1.EhPDI ExhibitsIn Vivo Isomerase Activity

To study the functional activities of eukaryotic PDI enzymesin vivo, yeast and bacterial cells have been successfully used to complement phenotypes associated with defective formation of disulfide bonds [32, 33]. InE. coli cells, the oxidative folding of polypeptides is carried out in the periplasmic compartment and performed by the Dsb proteins: oxidation and isomerization of disulfide bonds are catalyzed by DsbA and DsbC, respectively [34].The DsbC protein is particularly notable since it shares structural and functional similarities with eukaryotic PDI enzymes [35]. In fact, its functional role as disulfide isomerase has been studied using eukaryotic multidisulfide proteins as substrates [35, 36]. Four physiological substrates of DsbC have been identified so far: AppA [23], RcsF [37], MepA, and RNAse I [38]; from these,in vivo studies using AppA as substrate protein showed that DsbC plays an important role during folding of proteins with nonconsecutive disulfide bonds [23]. Then, to test whetherEhPDI exhibits disulfide isomerase activityin vivo, we performed a functional complementation assay using the ΔdsbC mutant ofE. coli as a model and the defective periplasmic expression of AppA as the phenotype.Initially, the absence of acid phosphatase activity (as background) was confirmed, indicating that the chromosomalappA gene was not induced under our experimental conditions (data not shown). Then, pBAD-AppA (Table 1) was used to transform both wild type and ΔdsbC mutant strains. Stable transformants were cultured and AppA expression was induced properly. As noted in Figure 1, a high level of acid phosphatase activity was detected in the wild type strain, 9595±593 Miller units, whereas a low level of activity was observed in the ΔdsbC mutant (1593±70 Miller units), confirming the DsbC-dependence of AppA (Figure 1). Then, pBPelB-EhPDI (Table 1) was used to transform the ΔdsbC/pBAD-AppA strain, using a two-plasmid system [39]. Stable cotransformants were cultured and protein expression was induced accordingly. Periplasmic expression ofEhPDI was confirmed by immunoblot (data not shown). Interestingly, a significant increase in acid phosphatase activity was detected (5151±344 Miller units), suggesting that the correct disulfide bond formation of AppA was assisted by the isomerase activity ofEhPDI.Figure 1
Acid phosphatase activity when AppA was expressed in the wild type and ΔdsbC mutant strains, as well as when it was coexpressed withEhPDI in the ΔdsbC mutant strain. The activity (expressed in Miller units) is shown on the left. The plasmids used for transfection ofE. coli are also indicated.


## 3.2. Isomerase Activity ofEhPDI Is Limited and Dependent on Its Reductase Activity

The active site of the thioredoxin-like domains from DsbA, DsbC, and PDI enzymes is characterized by the presence of the motif CXXC, where the cysteine residues play an important role in the enzymatic activity [40, 41].EhPDI contains two thioredoxin-like domains (referred to as N- and C-Trx, resp.; both having the motif CGHC) that are essential for itsin vivo oxidase activity [20]. To test whether both domains contribute to the isomerase activity, we carried out mutagenic analysis followed by a functional complementation assay.The pBluescript-based plasmids expressingEhPDI variants (Table 1) were used to transform the ΔdsbC/pBAD-AppA strain. Stable cotransformants were cultured and protein expression was induced accordingly. Periplasmic expression ofEhPDI variants was confirmed by immunoblot (data not shown). As indicated in Table 2, the isomerase activity ofEhPDI is dependent on its CGHC active sites, since a complete loss of the AppA activity was observed when the variant having both domains inactivated was coexpressed (EhPDISS/SS). Also, low AppA activity was detected when each domain was tested without the background of the other (EhPDISS/CC and EhPDICC/SS); furthermore, the slight difference observed between these two variants can be explained by considering that the thioredoxin-like domains are not equivalent with regard to the isomerase activity [33, 42]. However, it is important to take into account the cellular features of the bacterial model to better understand the role ofEhPDI as an isomerasein vivo.Table 2
In vivo isomerase activity of EhPDI enzyme variants.
Enzyme variant
Acid phosphatase (AppA)∗ activity in Miller units (%)¶
EhPDISS/SS
732 ± 74 (−1)
EhPDISS/CC
1086 ± 136 (7)
EhPDICC/SS
1646 ± 230 (20)
∗The data was expressed as ± standard error (n = 6).
¶Under the background of ΔdsbC/pBAD-AppA, normalization was performed considering the mean activity of pBPelB-EhPDI as maximal (Amax⁡ = 5151) and pBluescript SK- as minimal (Amin⁡ = 780). The percentage (%) was calculated as [(A-Amin⁡)/(Amax⁡-Amin⁡)] × 100, where A represents the activity of the enzyme variant.In the periplasmic compartment, substrate proteins with misoxidized disulfide bonds are shuffled to properly oxidized states by two mechanisms: (1) the isomerase pathway, where DsbC acts on the substrate as reductase-oxidase, and (2) the reductase pathway, where DsbC simply acts as reductase, allowing DsbA another chance to correctly oxidize the substrate [43]. In addition, when the ΔdsbC mutant ofE. coli was complemented with the protein TrxP fromBacteroides fragilis (a periplasmic reductase with poor isomerase activity), a fully restored AppA activity was observed, indicating that the disulfide bond isomerization of this substrate is accomplished mainly through the reductase pathway [43]. Hence, it is reasonable to think that the low AppA activity detected when the ΔdsbC mutant ofE. coli was complemented with any of the variants (Table 2) suggests that the isomerase function ofEhPDI is dependent on its reductase activity. To test this, we performed twoin vitro activity assays, oxidative refolding of ribonuclease and reduction of insulin, using purified recombinant enzymes, that is,EhPDI variants.As indicated in Table3, the wild type (EhPDI) and variants (EhPDISS/CC and EhPDICC/SS) showed comparable oxidative refolding capabilities. In contrast, significant differences were observed in their reductive activities: EhPDICC/SS retained about 60%, whilst EhPDISS/CC roughly retained 20%. These results confirmed thatEhPDI is dependent on its reductase activity to function as oxidoreductasein vivo andin vitro.Table 3
In vitro activities of purified EhPDI enzyme variants.
Enzyme
RNAse A oxidative refolding∗†¶
Insulin reduction∗†¶   [×10−6 A650/min2]  (%)
Oxidase[×10−6 min−1] (%)
Isomerase[×10−6 μM/min] (%)
EhPDI
513 ± 16 (100)
39 ± 1 (100)
109 ± 11 (100)
EhPDISS/CC
551 ± 16 (107)
33 ± 1 (85)
22 ± 2 (20)
EhPDICC/SS
517 ± 13 (101)
31 ± 1 (79)
65 ± 5 (60)
∗The data was expressed as mean ± standard error (n = 3).
†The activity of the enzyme variant EhPDISS/SS was not statistically significant, as compared with the reaction performed in the absence of enzyme.
¶The activity ratio (%) was calculated as (variant/wild type) × 100.


## 3.3.EhPDI Protects Proteins against Thermal-Induced Aggregation

The chaperone-like function of PDI enzymes is determined by their ability to protect misfolded/unfolded substrate proteins against thermal-induced aggregation and to assist refolding [44, 45]. This function is essential in order for PDI to act as an efficient folding catalyst, since it allows access to buried thiols and disulfide bonds in the substrates and prevents nonspecific interactions between partially folded intermediates [1]. Typically, the chaperone-like function has been studiedin vitro by measuring the ability to prevent protein aggregation induced by different physical or chemical conditions, such as temperature or denaturants [5, 46].We have already reported thatEhPDI exhibits chaperone-like function by showing its ability to prevent the DTT-induced aggregation of the B chain of insulin [21]. Although this assay was a simple approach to test the chaperone-like function ofEhPDI, the low molecular mass of the substrate (3.4 kDa) represents a limitation of this assay, since it offers a restricted number of contact sites to form stable complexes [47]. Hence, to gain further insights regarding the chaperone-like function ofEhPDI, we performed additionalin vitro assays to test its ability to prevent thermal-induced aggregation, using as substrates two heat-labile enzymes: α-glucosidase andNdeI endonuclease.As shown in Figures2 and 3, the chaperone-like function ofEhPDI was dose-dependent, since an increment of activity was observed as a result of augmenting its concentration. Moreover, to estimate its chaperone-like ability, the half-maximal effective concentration (EC50) and Hill slope were calculated by fitting the data to a model of one specific binding site with a variable slope. Interestingly, the apparent values obtained for α-glucosidase (EC50 = 3.0±0.4 μM; Hill slope = 1.0) andNdeI endonuclease (EC50 = 0.26±0.06 μM; Hill slope = 2.4) suggest thatEhPDI exhibits differences in substrate specificity and affinity [48].Figure 2
Protection of thermal inactivation ofα-glucosidase assisted byEhPDI. Relative chaperone-like activity (%) of recombinantEhPDI.Figure 3
Protection of thermal inactivation ofNdeI endonuclease assisted byEhPDI. Relative chaperone-like activity (%) of recombinantEhPDI. Upper panel: agarose gel indicating the relative mobility of the linearized and supercoiled plasmid (C1 and C2); also, concentrations ofEhPDI are indicated.AlthoughEhPDI does not have a substrate-binding b′-domain (a-a′-D) as the mammalian homologue (a-b-b′-a′-c) [49], the notion that some other domains (e.g., a′ or D) might be involved in the chaperone activity is evident. This idea is supported by the results of two previous reports: (i) ERp46, which lacks a b′-domain, is able to bind peptides through its catalytic domains (a0, a, and a′) [50] and (ii) the D-domain of ERp29 contains a discrete and conserved substrate-binding site [51].


## 3.4. Closing Remarks

Although little is known about theE. histolytica mechanisms that act in response to proteotoxic stress [52], the upregulation of genes encoding typical chaperones (such as Hsp70 and Hsp90) in a response to thermal stress suggests that it contains the cellular machinery necessary to preserve and restore the stability of the proteome [53]. So, the identification and characterization ofEhPDI as a folding catalyst with chaperone-like activity represents an additional step to dissect the molecular mechanisms involved in both protein folding and proteotoxic stress inE. histolytica. Hence, it is conceivable to suppose that inhibition ofEhPDI could lead to an increase in protein misfolding, promoting a sustained proteotoxic stress, eventually inducing apoptosis and, thus, preventing infection by this parasite.



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*Source: 286972-2015-01-28.xml*