An Atypical Protein Disulfide Isomerase from the Protozoan Parasite Leishmania Containing a Single Thioredoxin-like Domain*

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.
Protein disulfide isomerase (PDI) 1 (EC 5.3.4.1) is a member of the thioredoxin superfamily and is highly abundant in the lumen of the endoplasmic reticulum (ER) (1). PDI catalyzes the oxidation, reduction, and isomerization of disulfide bonds of proteins depending on the redox potentials in vitro (1) and is responsible in vivo for disulfide bond formation in nascent polypeptides in the ER (2,3). In addition to its redox/isomerase activities, PDI has been shown to have chaperone activity. For example, PDI facilitates the refolding of denatured lysozyme (4) and acid phospholipase A2 (5) as well as denatured proteins that lack disulfide bridges such as glyceraldehyde-3-phosphate dehydrogenase (6) and rhodanese (7). According to a recent models, a "typical" PDI protein is composed of four consecutive thioredoxin-like domains (a-b-bЈ-aЈ) of which only two, a and aЈ, contain the active site -CGHC- (8). These four thioredoxin domains are flanked by an N-terminal signal peptide to permit translocation of the protein into the ER and a C-terminal c domain that is rich in acidic amino acids and contains the KEDL (or KEDL-like) retention signal (8,9). Even though most of the PDIs described to date agree with this five-domain structure model, there is an increasing number of proteins, belonging to the PDI family, that do not follow that model (8). Among those "atypical" PDIs are ERP-57, ERP72, or ERp28, which show differences in the number and organization of their thioredoxin domains (8). Despite some heterogeneity in structure, it is believed that most members of the PDI family must fulfill both enzymatic and chaperone functions in the ER of eukaryotic cells (8).
To date, little is known about the structure and the role of PDIs in lower eukaryotes such as the protozoan parasites. A typical ϳ55-kDa PDI (containing two -CGHC-active sites and four thioredoxin domains) has been identified in two human trypanosomatid parasites Trypanosoma brucei (10) (causing sleeping sickness) and Leishmania major (11) (causing cutaneous leishmaniasis). Only the L. major PDI was shown to possess redox/isomerase activities in vitro (11). In addition, several smaller size PDI proteins (12-50 kDa) have also been identified in the early branching protozoan eukaryote Giardia lamblia (12,13). One of them (G. lamblia PDI-3), containing only one thioredoxin domain with an active site, was shown to possess oxidase and isomerase activities in vitro (12). The biological function of PDI in the protozoan parasites remains to be elucidated. However, recently an increased expression of PDI in a highly virulent strain of L. major was demonstrated, thereby suggesting that PDI may have a role in Leishmania pathogenesis (11). The understanding of the structure and function of PDIs in lower eukaryotes is of significant interest to better understand the phylogeny of the PDI family (13) and elucidate the role of PDIs as ER chaperones involved in the quality control of the folding and secretion of virulence factors produced by protozoan parasites such as Leishmania (14).
Leishmania are trypanosomatid parasites responsible for a spectrum of human diseases ranging from mild cutaneous to often fatal visceral leishmaniasis (15). During its life cycle, the parasite alternates between extracellular promastigotes in the gut of the sand fly insect vector and obligatory intracellular * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The amastigotes within the phagolysosomal compartment of macrophages in vertebrate hosts (15). Some Leishmania virulence factors include both surface membrane-bound and -secreted molecules that play a role in the parasite survival such as evading killing by the host immune system (16,17) or acquiring essential nutrients from the host (18 -20). It has been shown that Leishmania proteins bound for secretion (i.e. secretory acid phosphatase, SAcP) (21) follow a typical eukaryotic secretory pathway, i.e. traveling from the ER to the Golgi apparatus to the cell surface, or are secreted via vesicular trafficking through a flagellar reservoir, which is the only site of exocytosis in these parasites (22). Therefore, as in higher eukaryotes (23,24) the ER chaperones such as PDI could play a critical role in the control of folding and secretion of leishmanial proteins to ensure their critical functions in parasite survival. In that regard, we have recently reported that the disruption of the leishmanial ER chaperone calreticulin function resulted in a significant decrease in the secretion of one of the major secretory proteins, secretory acid phosphatase, which correlates with reduced parasite survival in macrophages (14). In this report we identified and characterized for the first time in trypanosomatid parasites an atypical, single thioredoxin-like domain containing PDI from Leishmania donovani. We showed that this protein is enzymatically active and is localized in the ER of the parasite. Furthermore, altering the activity of this PDI affects the secretion of the major L. donovani secretory protein, thereby suggesting that it may play a role in the parasite secretory pathway.
Reverse Transcriptase Polymerase Chain Reaction and Cosmid Library Screening-Messenger RNA was purified from total RNA using a mRNA isolation kit (Ambion, Austin, Texas) and used in an reverse transcription reaction using hexanucleotides as primers and the reverse transcriptase RTII (Stratagene). To identify putative PDI genes, this cDNA was used as a template in a polymerase chain reaction with the forward 5Ј-ACT AAC GCT ATA TAA GTA TCA-3Ј and reverse 5Ј-TT (A/G)CA (A/G)TG (A/G/C)CC (A/G)CA CCA-3Ј primers. The forward primer reflects a portion of the conserved Leishmania-specific mRNA splice leader sequence (27), and the degenerate reverse primer is based on conserved amino acids, including the active site, of various PDI sequences available in protein databases. The resulting PCR products were cloned into the pCRII cloning vector (Invitrogen) and sequenced. A positive clone containing a PCR fragment that showed a high sequence homology with PDIs by BLAST analysis was selected. The nucleotide sequence corresponding to the PDI open reading frame of this positive clone was further amplified by PCR, using appropriate primers, and used as a probe to screen an L. donovani cosmid library as described previously (25). A positive cosmid clone (cosmid 10-3) was isolated and sequenced in the region of the putative LdPDI gene.
Northern and Southern Blot Analyses-Total RNA was extracted from L. donovani promastigotes and axenic amastigotes using RNA STAT-60 (TEL-TEST, Inc.) and according to the manufacturer's instructions. Genomic DNA was isolated from L. donovani promastigotes according to the methods described in the manual for the genome DNA isolation kit from BIO 101, Inc. Southern and Northern blot hybridizations as well as the preparation of the radiolabeled LdPDI nucleotide probes were carried out according to standard protocols (28).

Leishmania, and Bacteria Expression Plasmid Constructs
pKS NEO LdPDI-wt-The wild type LdPDI gene was amplified by PCR using the LdPDI-containing cosmid (cosmid 10-3) described above as a template and the following forward primer-1 and reverse primer-2. Primer-1 was 5Ј-GG ACT AGT ATG TCC CTC GTC CGG AAG-3Ј, which contains a SpeI restriction site (bold) and the first 18 nucleotides of the LdPDI gene sequence (start codon is underlined). Primer-2 was 5Ј-CC ACT AGT CTA cgc gta gtc cgg cac gtc gta cgg gta CTG CTT GTT GGC CGC CAC-3Ј, which contains, sequentially, a SpeI restriction site (bold), a stop codon (underlined), a sequence for an hemagglutinin (HA) tag (lowercase), and 18 nucleotides of LdPDI 3Ј-end gene sequence. The resulting PCR product was initially cloned into the pCRII cloning vector (T/A cloning system, Invitrogen). The fidelity of the cloned sequence was verified by nucleotide sequencing. The SpeI insert was subsequently cloned into the SpeI site of the pKS NEO Leishmania expression plasmid (29) to generate the pKS NEO LdPDI-wt expression plasmid. The orientation of the SpeI fragment in pKS NEO was verified by digestion with the appropriate restriction enzymes.
pKS NEO LdPDI-mut-A portion of the LdPDI gene was first amplified by PCR using pKS NEO LdPDI-wt as a template and the forward primer-1 described above and the following reverse primer-3. Primer-3 was 5Ј-CTC CAG CCA CGT CGG CTT CAT GTT GTT GGC GTG GCC GGC CCA CGG AGC-3Ј, which corresponds to an internal LdPDI sequence that contains a BglI restriction site (bold) and two modified codons (underlined) that substitute the two cysteins of the LdPDI active site with alanine residues. The resulting PCR product was initially cloned into pCRII, and the EcoRI/BglI fragment isolated from that plasmid was subsequently ligated with the BglI/SpeI fragment of the pKS NEO LdPDI-wt plasmid. The resulting ligation reaction was subsequently used as template in a PCR with primers 1 and 2 to amplify the complete LdPDI gene containing mutated codons. The resulting PCR product was initially cloned into the pCRII plasmid and sequenced, and the SpeI insert from that plasmid was ligated into the SpeI site of the pKS NEO to generate the pKS NEO LdPDI-mut expression plasmid. The orientation of the SpeI fragment in pKS NEO was verified by digestion with appropriate restriction enzymes.
Escherichia coli-recombinant LdPDI Proteins and Antibody Production-An E. coli expression plasmid containing the wild type LdPDI gene was made. To that end, a portion of the LdPDI gene lacking a sequence encoding the putative signal peptide was amplified by PCR using the LdPDI-containing plasmid pKS NEO LdPDI-wt as template and the following forward primer-4 and reverse primer-5. Primer-4 was 5Ј-GGC ATG CAG gag gtg gtc gag ctc aac-3Ј, which contains an SphI restriction site (bold) followed by 18 nucleotides of the LdPDI gene sequence (lowercase). Primer-5 was 5Ј-AGA TCT ctg ctt gtt ggc cgc cac-3Ј, which contains a BglII restriction site (bold) followed by the last 18 nucleotides of the LdPDI gene sequence (excluding the stop codon, lowercase). The resulting PCR product was digested with SphI and BglII enzymes, purified, and ligated into the SphI/BglII site of the pQE-70 expression plasmid (Qiagen). The resulting expression plasmid encodes the LdPDI protein containing a C-terminal His 6 tag (LdPDI-His). A second expression plasmid encoding LdPDI in which the -CGHC-active site had been mutated to -AGHA-was also made. To that end, the mutated LdPDI sequence was amplified by PCR using pKS NEO LdPDI-mut as template and primers 4 and 5. The resulting PCR fragment was prepared for expression in pQE-70 as above. The M15 E. coli host cells (Qiagen) were transformed with these two plasmids. The purification under non-denaturing conditions of either the LdPDI-His or the LdPDI-mut-His proteins was performed from selected clones and according to Qiagen protocols. Furthermore, the purified LdPDI-His protein was used to immunize a New Zealand White rabbit according to company protocol (Spring Valley Laboratories). The resulting antiserum (anti-LdPDI) was shown to specifically react against the parasite LdPDI by Western blot.
Analysis SDS-PAGE and Western Blotting-L. donovani promastigotes were harvested by centrifugation (1,500 ϫ g for 10 min), washed in phosphate buffer saline (PBS), and lysed in SDS-PAGE sample buffer. Proteins from an equivalent number of cells (2-4 ϫ 10 6 cells) were analyzed by SDS-PAGE, transferred onto nitrocellulose, and processed for Western blot analysis with the various antibodies as described previously (25).
PDI Enzymatic Assays-Purified E. coli recombinant LdPDI-His or mutant LdPDI-mut-His were assayed for PDI enzymatic activities. The ability of recombinant proteins to catalyze the refolding of "scrambled" bovine pancreatic ribonuclease type II-A (Sigma; a measure of disulfide isomerization) was determined as described by Hawkins et al. (30). The PDI-catalyzed folding of reduced bovine pancreatic trypsin inhibitor (BPTI; type I-P, Sigma; a measure of oxidative formation of native disulfide bonds) was measured as described by Creighton  Rat liver PDI, purified according to the method of Lambert and Freedman (32), was used as positive control in these assays.
Immunofluorescence-L. donovani promastigotes were fixed in suspension in 4% (w/v) paraformaldehyde (Sigma) in PBS for 20 min on ice, washed three times in PBS, added to glass slides, and air dried. Cells were permeabilized in absolute methanol at Ϫ20°C for 6 min, rinsed in PBS, and incubated for 30 min in PBS containing 5% (w/v) bovine serum albumin (United States Biochemical). Subsequently, cells were incubated for 1 h with either the anti-LdPDI or the anti-immunoglobulin heavy chain binding protein (BiP) (kindly provided by J. Bangs, University of Wisconsin, Madison, WI) antibodies for single staining, and for double staining they were incubated with a mixture of anti-BiP and anti-hemagglutinin tag epitope (Roche Molecular Biochemicals) antibodies at appropriate dilutions in 1% (w/v) bovine serum albumin in PBS. Following three washes in PBS, slides were incubated for 1 h with either fluorescein goat anti-rabbit or with a mixture of rhodamineconjugated goat anti-rat and fluorescein-conjugated rabbit anti-rabbit antibodies (1/200 dilution) (Vector Laboratories) diluted in 1% (w/v) bovine serum albumin in PBS. Following three washes in PBS, slides were mounted in Vectashield (Vector Laboratories) and examined with a Carl Zeiss laser-scanning confocal microscope (model LSM5 PASCAL) equipped with a microprocessor. The images were further processed using Adobe Photoshop 5.5 (Adobe Systems).
SAcP Enzymatic Assay-Cultures of promastigote transfectants were centrifuged at 1,500 ϫ g for 10 min at 4°C. Culture supernatants were harvested and further centrifuged at 10,000 ϫ g for 15 min at 4°C to eliminate remaining cellular debris. Cleared culture supernatants were assayed for acid phosphatase activity using paranitrophenyl phos-phate (Sigma) as a substrate as described previously (18). Acid phosphatase enzymatic activity is expressed as a nanomole of the substrate hydrolyzed per minute per 10 7 cells (nmol/min/10 7 cells). Student's t test was used to determine significance.

RESULTS
Isolation of a Putative PDI Gene from L. donovani-A gene encoding a putative PDI has been cloned from the protozoan parasite L. donovani (Fig. 1). The 5Ј-region of the gene was first amplified by reverse transcriptase PCR using total RNA isolated from L. donovani promastigotes as the template and the following 5Ј-and 3Ј-primers. The 5Ј-forward primer was based on the spliced leader sequence, which is added to all leishmanial mRNAs by transplicing (27). The 3Ј-reverse degenerate primer was deduced from a region containing the highly conserved active site -CGHC-motif of PDIs (8,9). A ϳ300-bp PCR fragment was obtained that contained an open reading frame showing homology with PDIs (data not shown). This PCR fragment was therefore used as a specific probe to screen a L. donovani cosmid library (14). From such a screening, a cosmid clone with a 402-bp open reading frame encoding a 133-amino acid protein was identified (Fig. 1B). The open reading frame was 61% GC rich, which agrees with the GC content of the Leishmania genome (33). Based on the sequencing re- sults of the reverse transcriptase-PCR fragment described above, the spliced leader addition site was localized 108 nucleotides upstream to the start codon (Fig. 1C). The deduced protein sequence from the complete open reading frame showed high sequence similarity with PDI from other organisms by BLAST analysis (not shown). It also contains a 21-amino acid N-terminal putative signal peptide (Fig. 1B, underlined) which could facilitate the translocation of the nascent protein into the ER. The deduced protein also contains the typical -CGHC-PDI signature (Fig. 1B, box) corresponding to the active site of this family of enzymes (8,9). Based on these basic characteristics, we concluded that the cloned gene corresponds to a putative PDI gene in Leishmania, and hence we designated it Leishmania donovani PDI (LdPDI).
Structural Comparison of the Putative LdPDI with Other PDIs-PDI is a member of the superfamily of protein-thiol oxidoreductase enzymes with sequence and structural similarity to thioredoxin (9). The schematic diagram showing the structure of a typical PDI is shown in Fig. 1A. It is comprised of two domains, a and aЈ, having high sequence similarity with thioredoxins, and each contains one -CGHC-active site separated by two thioredoxin-related domains, b and bЈ, and followed by the calcium binding c domain containing a C-terminal KDEL ER retention signal (8,9). Unlike a typical ϳ55-kDa PDI protein, the L. donovani putative LdPDI gene encodes for a ϳ12-kDa protein. Clustal sequence alignment of LdPDI with PDIs from other organisms shows that it has homology to the a domain of PDIs with a block of conserved amino acids in the region of the -CGHC-active site (Fig. 1B). A second block of conserved amino acids, G(Y/F)PT, was also observed at position 98 -101 of the L. donovani sequence (Fig. 1B). In addition to its small size, the LdPDI does not contain either the acidic Cterminal domain or an ER retention signal as is found in most PDIs (8). Recently, a typical four-thioredoxin-like domain PDI has been reported in L. major, a related Leishmania species (11). However, the putative LdPDI protein shows only 44% similarity and 35% identity with the L. major PDI. These results suggest the presence of a novel single thioredoxin-like domain PDI in Leishmania.
Genomic Organization and Expression of the Putative LdPDI Gene-To define the complexity of the LdPDI gene in the Leishmania genome, L. donovani genomic DNA was digested with several restriction enzymes selected to demonstrate genome copy number and hybridized at a high stringency with the LdPDI gene probe in Southern blot analysis. The probe hybridized with a single band when genomic DNA was digested with PstI or XhoI, which were selected to cut outside the probe sequence ( Fig. 2A, lanes 1 and 3). However, two fragments hybridized with the probe when DNA was digested with either NcoI or SacII, which were selected to cut once within the probe sequence ( Fig. 2A, lanes 2 and 4). In addition, double digestion of genomic DNA either with PstI and NcoI or with XhoI and NcoI also resulted in two fragments ( Fig. 2A, lanes 5 and 6). This result suggests that the putative LdPDI gene is present as a single copy in L. donovani. Of interest in these Southern blot experiments performed under high stringency, several weak bands hybridizing with the LdPDI gene probe were also observed ( Fig. 2A, asterisks). These could reflect hybridization with other related genes such as thioredoxin genes or other member of the PDI family, which would have significant sequence homology with the cloned putative LdPDI gene. However, this observation requires further investigation. Expression of the LdPDI gene as determined by Northern blot analysis showed that the putative LdPDI gene was transcribed equally in both life stages of the L. donovani parasites, because the LdPDI gene probe hybridized equally with a single ϳ1.5-kb transcript in both promastigotes and axenic amastigotes (Fig. 2B, lanes 1 and 2, respectively).
Enzymatic Activity of the Recombinant LdPDI Protein-To determine whether the putative LdPDI gene encodes for an active PDI enzyme, the protein was expressed in E. coli and assayed for PDI enzymatic activities. As a control in these experiments, a mutant form of the putative LdPDI was also expressed in E. coli. This mutant form only differs from the wild type by having the two cysteine residues of its -CGHCactive site replaced by two alanine residues. Such a mutation should result in a complete loss of enzyme activity as previously shown for the human PDI (34). Both the wild type (LdPDI-His) and mutant (LdPDI-mut-His) forms of LdPDI were expressed as histidine-tagged proteins and purified from E. coli lysates under non-denaturing conditions. SDS-PAGE analysis of the purified proteins stained with Coomassie Blue is shown in Fig. 3A. Both LdPDI-His and LdPDImut-His have an apparent molecular mass of ϳ12 kDa under these conditions (Fig. 3A).
PDI is a member of the protein thiol-disulfide oxidoreductase family, capable of catalyzing both oxidation and reduction of protein disulfides (9). The catalysis of disulfide bond formation (or oxidation) by PDI can be quantitated by measuring the refolding of reduced BPTI as it regains the ability to inhibit trypsin hydrolysis (31). LdPDI-His and LdPDI-mut-His were subjected to such an assay in which the rat PDI was used as positive control. Like the rat PDI, the LdPDI-His showed a time-dependant increase in BPTI folding, whereas LdPDI-mut-His and negative controls had very low or negligible activity (Fig. 3B). PDI enzymes also have the ability to refold RNase-A that has been reduced, denatured, and randomly refolded by reoxidation in air (scrambled). This assay has been used as a common measure of protein-disulfide isomerization (30). In this assay, LdPDI-His showed significant activity in refolding the scrambled RNase-A, similar to the rat PDI, whereas Ld-PDI-mut-His showed no activity (Fig. 3C). Taken together, these results demonstrate that the recombinant LdPDI-His is able to catalyze both oxidation and isomerization of protein disulfides and that the -CGHC-active site is indeed required for its enzymatic activities. These results demonstrate that LdPDI gene encodes for an active PDI enzyme.
Localization of LdPDI in L. donovani Promastigotes-Because the LdPDI does not have a typical C-terminal ER retention sequence, it was important to determine its cellular localization in the parasite. To that end, a LdPDI-specific rabbit antiserum was generated against the purified histidine-tagged (LdPDI-His) protein described above. This specific anti-LdPDI antibody was used in indirect immunofluorescence assays to localize LdPDI in L. donovani promastigotes. Results showed that the anti-LdPDI antibody reacted with proteins localized in a reticular network in the entire body of the promastigote, excluding the nucleus (Fig. 4 panel B). The fluorescence was stronger at the periphery of the nucleus. This pattern of fluorescence was similar to that of BiP, another ER resident protein ( Fig. 4 panel C). The anti-BiP antibody used in this experiment is specific to T. brucei and was shown to localize the BiP protein in the endoplasmic reticulum of this parasite (35), as well as in the related trypanosomatid parasites Trypanosoma cruzi (36) and Leishmania mexicana amazonensis (37). No fluorescence was seen in promastigotes treated with a normal rabbit serum (Fig. 4, panel A). Because the fluorescence patterns observed by indirect immunofluorescence assay with the anti-BiP and the anti-LdPDI antibodies are very similar, we concluded that the LdPDI was localized at least in part in the ER of L. donovani.
Overexpression of LdPDI in L. donovani Promastigotes-To assess the role of LdPDI in the secretory pathway of L. donovani, transfected parasite cells lines expressing either a wild type (LdPDI-wt) or mutant inactive (LdPDI-mut) form of Ld-PDI were generated. The culture supernatants of these transfected parasites were then assayed for SAcP activity (18). The SAcPs were used as marker proteins for secretion because they represent the major secreted glycoproteins by L. donovani, and their trafficking through the parasite secretory pathway has been well documented (21,38).
First, both the wild type (-CGHC-) and a mutant/inactive (-AGHA-) form of LdPDI were episomally expressed as HAtagged proteins (Fig. 5A) in transfected parasites using the pKS NEO Leishmania expression plasmid (29). The expression of the HA-tagged proteins by the transfected parasites was first confirmed by Western blot analysis (Fig. 5B). Results showed that the anti-HA antibody reacted with a ϳ13-kDa protein in lysates of both LdPDI-wt and LdPDI-mut (Fig. 5B, lanes 2 and  3, respectively) and gave only background reactivity with lysates of control LdKS (Fig. 5B, lane 1). The control cell line (LdKS) was transfected with the expression plasmid alone. Similarly, cell lysates were reacted with the anti-LdPDI-specific antibody described above. This antibody reacted with a ϳ13-kDa protein in lysates of LdPDI-wt and LdPDI-mut (Fig.  5B, lanes 5 and 6), whereas the control pre-immune rabbit serum showed only background reactivity in lysates of control LdKS (Fig. 5B, lane 7). In addition to the ϳ13 kDa episomally expressed PDI, the anti-LdPDI-specific antibody also reacted with a ϳ12-kDa protein in lysates of the three cell lines LdKS, LdPDI-wt, and LdPDI-mut (Fig. 5B, lanes 4 -6), demonstrating the expression of the endogenous LdPDI in L. donovani promastigotes. Such expression was also observed in wild type promastigotes as well as in axenic amastigotes of L. donovani (data not shown). Furthermore, the expressed LdPDI-wt and LdPDI-mut HA-tagged proteins were shown to co-localize with BiP in transfected LdPDI-wt and LdPDI-mut parasites by confocal microscopy (Fig. 5C), thereby suggesting that these two episomally expressed proteins were properly targeted to the ER of transfected parasites. These studies demonstrated the expression and proper localization of both the wild type and mutant forms of LdPDI in L. donovani transfectants.
Next, to measure the release of SAcPs by these transfected parasites, supernatants of LdKS, LdPDI-wt and LdPDI-mut cultures were harvested on various days after inoculation with the same number of parasites and assayed for acid phosphatase enzymatic activity. The three cell types did not show significant differences in their growth rate in culture (Fig. 6A). However, at the end of the log phase of growth (96 h), culture supernatants of LdPDI-mut contained significantly (p ϭ 0.05) less (30%) secreted acid phosphatase activity than supernatants of LdKS controls or LdPDI-wt (Fig. 6B). No difference in the amount of acid phosphatase activity secreted by LdKS and LdPDI-wt was found (Fig. 6B). These results show that overexpression of an inactive form of LdPDI by the parasite results in a significant decrease in their secretion of SAcP. However, overexpression of the wild type PDI did not have any deleterious effects on the secretion process. DISCUSSION In this study we showed that the lower eukaryote Leishmania possesses a small (ϳ12 kDa) single thioredoxin-like domain containing PDI. Sequence alignment of LdPDI with other members of the PDI family revealed that it contains a characteristic -CGHC-PDI active site. The PDI -CGHC-active site is different from the conserved -CGPC-active site found in all thioredoxin enzymes (8), thereby suggesting that LdPDI belongs to the PDI family and not to the thioredoxin family. In addition, PDI sequence alignment showed that the LdPDI cloned in this study was homologous to the "a" domain of the archetype PDI that contains four thioredoxin-like domains and an apparent molecular mass of ϳ55 kDa (8,9). The LdPDI is the first single thioredoxin-like domain containing PDI to have been characterized in the trypanosomatid family of organisms. To date, PDIs containing a single active site have been reported only in bacteria (Dsb family of proteins) (39), yeast (40), fungi (41), and from the protist G. lamblia (12,13). The finding of a single thioredoxin-like domain PDI in Leishmania supports the hypothesis of McArthur et al. (13) who suggested that lineages of single-domain PDI have existed since the origin of eukaryotes and persist throughout eukaryotic diversity. Because most organisms studied to date have more than one PDI or PDI-related gene (42), it is likely that L. donovani possess additional PDIs. Our Southern blot results support the existence of PDI-related genes in this parasite, because minor bands of hybridization with the LdPDI gene probe were observed in our experiments ( Fig. 2A). Recently, a typical four-thioredoxin-like domain PDI has been reported in L. major, a related Leishmania species (11). Furthermore, three independent partial PDI gene sequences from L. major are also available in genome survey sequence data base (accession numbers AQ902241, AQ849985, and AQ911533). These observations suggest that L. donovani may also possess several PDI genes or PDI-related genes. However, these PDI-related proteins probably have low antigenic similarity with the LdPDI reported in this study, because the anti-LdPDI antibody reacted only with a single ϳ12kDa protein in lysates of L. donovani by Western blots.
Although PDI is mainly localized in the ER, it has also been found in other intracellular compartments such as the plasma membrane, the cytosol, and, in some cases, it is also secreted (8,9). It is clear from our indirect immunofluorescence assay studies that the LdPDI is mainly localized in the ER of the parasites. However, the LdPDI protein does not have the typical KDEL (or KDEL-like) ER retention signal that has been found in other leishmanial ER resident proteins (e.g. KEDL in calreticulin and MDDL in GRP78 (43,44), respectively). The mechanism by which the LdPDI is retained in the parasite ER is not known at this point. It is possible that Leishmania has additional mechanisms by which proteins, such as LdPDI, can be retained in the ER. Alternatively, the ER localization of LdPDI could be achieved via protein-protein interactions with other ER-resident proteins. In higher eukaryotes, PDI was shown to interact strongly with calreticulin and to be a part of multiprotein complexes in the ER including BiP, calreticulin, and ERP57 (45,46). Similar interaction between LdPDI and other leishmanial ER resident proteins could explain its ER localization as observed in this study. The primary role of PDIs is to catalyze the oxidation and isomerization of protein disulfide bonds in the ER (8,9). The catalytic mechanism for the formation or reduction of disulfides by PDI has been well characterized (8,47). The catalytic site in PDI involves the -CGHC-amino acid residues found in the thioredoxin domains a and aЈ of the enzyme (48). It was shown previously that the a and aЈ domains of PDI, when expressed alone, both act as reductases and oxidases, depending on the substrate and the redox environment (49). However, the recombinant a and aЈ domains, when expressed alone, are not able to catalyze the isomerization of complex substrates in vitro (e.g. BPTI) (50). Only the full-length PDI is able to catalyze the isomerization of such complex substrates (51). In contrast, our study shows that the LdPDI, which has only one thioredoxin domain, is able to catalyze isomerization such as the refolding of BPTI in vitro. Similarly, other single domain PDIs from the protist G. lamblia were also shown to catalyze the refolding of BPTI in vitro (12). Taken together, these studies suggest that, in evolution, single thioredoxin domain PDIs could act as a functionally active isomerases, such as in the unicellular organisms Leishmania and Giardia. However, in multicellular organisms the association of multiple thioredoxin-like domains is required to form a functionally active PDI. Such multidomain PDIs could have various functions, including isomerase and chaperone activities. Furthermore, a single thioredoxin domain PDI could be the ancestral form of PDI that, by gene duplication, produced PDI diversity to perform other related functions (13).
Despite heterogeneity in size and domain organization, it is believed that most members of the PDI family function both as oxidoreductases/isomerases and also as molecular chaperones in the ER of eukaryotic cells (8). In higher eukaryotes, ER chaperones are part of the ER quality control machinery that regulates the folding and secretion of cell surface-anchored and -secreted proteins (23). To date, only a few homologues of ER chaperones of higher eukaryotes have been isolated in Leishmania (11,43,44,52,53). With the exception of GRP94 (53), the direct involvement of these proteins in the parasite secretory pathway has not been demonstrated. Our current results suggest the single thioredoxin-like domain LdPDI plays a role in the secretory pathway of L. donovani, because the overexpression of an inactive form of LdPDI (LdPDI-mut) significantly reduced the secretion of one of the major secretory proteins, secretory acid phosphatase. These results suggest a dominant negative interaction between SAcP and the inactive LdPDI-mut, resulting in incorrect disulfide bond formation and probably incorrect folding and degradation of the SAcP enzymes. Alternatively, the mutant/inactive LdPDI-mut could compete with the endogenous LdPDI in the formation of multichaperone folding complexes in the ER, resulting in inefficient complexes unable to catalyze the folding and assembly of proteins trafficking through the parasite secretory pathway (e.g. SAcPs). The exact mechanisms involved in the reduced secretion of SAcP by parasites expressing a mutant/inactive LdPDI are currently being explored.
In conclusion, we have isolated and characterized a functional single thioredoxin-like domain PDI for the first time in a trypanosomatid parasite. Furthermore, we have shown that disruption of PDI activity in Leishmania can result in an alteration of the secretion of parasite secretory proteins. Because many secreted or cell surface-anchored proteins of Leishmania represent important parasite virulence factors, altering the function of ER resident proteins could be exploited to attenuate the parasite virulence in order to develop live vaccine against human leishmaniasis.