From the Divisions of Emerging and Transfusion
Transmitted Diseases and
Viral Products, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda,
Maryland 20892 and the ¶ Division of Basic Biomedical Sciences,
University of South Dakota School of Medicine,
Vermillon, South Dakota 57069
Received for publication, October 9, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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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-b'-a') of which only two, a and a', 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 ~55-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 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.
Parasite Cultures and Transfections--
L. donovani
promastigotes (strain 1S, clone 2D, World Health Organization
designation MHOM/S.D./62/1S-CL2D) (25) were grown in M199 medium
containing 10% heat-inactivated fetal bovine serum and harvested by
centrifugation as described previously (25). Axenic amastigotes of
L. donovani were derived from promastigotes as described
previously (26). Promastigotes were transfected by electroporation and
selected for growth in medium containing Geneticin (G418) up to 400 µg/ml, as described previously (20).
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 5'-ACT AAC GCT ATA TAA GTA TCA-3' and reverse 5'-TT (A/G)CA
(A/G)TG (A/G/C)CC (A/G)CA CCA-3' 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 5'-GG ACT AGT
ATG TCC CTC GTC CGG AAG-3', which contains a
SpeI restriction site (bold) and the first 18 nucleotides of the LdPDI gene sequence (start codon is underlined).
Primer-2 was 5'-CC ACT AGT CTA cgc gta gtc cgg
cac gtc gta cgg gta CTG CTT GTT GGC CGC CAC-3', 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 3'-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 5'-CTC CAG CCA CGT CGG CTT CAT GTT GTT
GGC GTG GCC GGC CCA CGG AGC-3', 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
5'-GGC ATG CAG gag gtg gtc gag ctc aac-3', which contains an
SphI restriction site (bold) followed by 18 nucleotides of
the LdPDI gene sequence (lowercase). Primer-5 was 5'-AGA TCT ctg ctt gtt ggc cgc cac-3', 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 His6 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 × 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 × 106 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 et al. (31) in 0.2 M Tris-Cl containing glutathion (0.1 mM GSSG
and 0.2 mM GSH). 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 SAcP Enzymatic Assay--
Cultures of promastigote transfectants
were centrifuged at 1,500 × g for 10 min at 4 °C.
Culture supernatants were harvested and further centrifuged at
10,000 × g for 15 min at 4 °C to eliminate remaining cellular debris. Cleared culture supernatants were assayed for acid phosphatase activity using paranitrophenyl phosphate (Sigma)
as a substrate as described previously (18). Acid phosphatase enzymatic
activity is expressed as a nanomole of the substrate hydrolyzed per
minute per 107 cells (nmol/min/107 cells).
Student's t test was used to determine significance.
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 5'-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 5'- and 3'-primers. The 5'-forward
primer was based on the spliced leader sequence, which is added to all
leishmanial mRNAs by transplicing (27). The 3'-reverse degenerate
primer was deduced from a region containing the highly conserved active site -CGHC- motif of PDIs (8, 9). A ~300-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 results 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 a', having high sequence similarity with
thioredoxins, and each contains one -CGHC- active site separated by two
thioredoxin-related domains, b and b', and followed by the calcium
binding c domain containing a C-terminal KDEL ER retention signal (8,
9). Unlike a typical ~55-kDa PDI protein, the L. donovani
putative LdPDI gene encodes for a ~12-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 C-terminal 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 ~1.5-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 -CGHC- active 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
LdPDI-mut-His have an apparent molecular mass of ~12 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 LdPDI-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 LdPDI 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 HA-tagged 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 ~13-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 ~13-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 ~13 kDa episomally expressed PDI, the
anti-LdPDI-specific antibody also reacted with a ~12-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 = 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.
In this study we showed that the lower eukaryote
Leishmania possesses a small (~12 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 ~55 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
~12kDa 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 a' of the
enzyme (48). It was shown previously that the a and a' 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 a' 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °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 rhodamine-conjugated 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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (86K):
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Fig. 1.
Characteristics of the L. donovani
PDI. A, top, schematic representation
of a typical PDI containing an N-terminal signal peptide
(SP, black box), a C-terminal KDEL ER
retention signal, and the five characteristic domains of PDI,
i.e. a, b, b', a', and c. The -CGHC- active sites present in
domains a and a' are indicated (hatched box).
Bottom, map of the LdPDI containing a putative N-terminal
signal peptide (SP, black box) and a single
-CGHC- active site (hatched box). B, multiple
sequence alignment of LdPDI (GenBankTM accession number
AY166592) with PDI proteins from various eukaryotes species: Homo
sapiens (P07237), Zea mays (P52588), Drosophila
melanogaster (AAA86480), Schistosoma japonicum
(AAC78302), Saccharomyces cerevisiae (AAA34848), and
Giardia intestinalis isoform 3 (AAF20171) and isoform 5 (AAF89535). The gray boxes represent identical
amino acids. The putative signal peptide sequence of the LdPDI is
underlined, and the conserved -CGHC- active site of the PDIs
are boxed. Alignment of the first ~180 amino acids is
shown. C, results of reverse transcription PCR showing the
5'untranslated region of the LdPDI gene. The first
nucleotide of the LdPDI open reading frame is indicated (1).
The splice leader sequence is underlined, and the
arrow indicates the splice leader acceptor site. The first
five amino acid residues of the LdPDI protein are shown
(uppercase).
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Fig. 2.
Southern and Northern blot analyses of
L. donovani PDI. A,
Southern blot. L. donovani genomic DNA (5 µg) was digested
with the restriction enzymes indicated at top (P,
PstI; N, NcoI; X,
XhoI; S, SacII), separated on 1%
agarose gel, transferred to nylon membrane, and hybridized with the
LdPDI gene as a probe. Asterisks indicate minor
bands of hybridization. B, Northern blot. 10 µg of total
RNA from each of the mid-log parasites (P, promastigote;
A, axenic amastigote) was separated in an
agarose/formaldehyde gel and hybridized to the LdPDI gene as
a probe. Molecular weight standards (in kb) are indicated on the
left of both the panels.
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Fig. 3.
Recombinant L. donovani PDI
shows PDI enzymatic activities in vitro.
A, SDS-PAGE of the nickel-agarose purified, E. coli-expressed, recombinant LdPDI proteins stained with Coomassie
Blue. LdPDI proteins (250 ng) of either the wild type (WT)
containing the -CGHC- active site or mutant (Mut) containing
the mutated -AGHA- site, were analyzed. Molecular mass of protein
standards (in kDa) are shown on the left. B,
Catalysis of folding of reduced and denatured BPTI as a measure of
oxidative formation of disulfide bonds by LdPDI-His, LdPDI-mut-His, and
rat PDI used as positive control and compared with the no enzyme
control. Results are expressed as a percentage of BPTI refolded.
C, catalysis of refolding of scrambled ribonuclease A as a
measure of disulfide isomerization by the same samples as in
panel B. The data shown here are from one
experiment that is representative of two to three separate
experiments.
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Fig. 4.
Immunolocalization of LdPDI in L. donovani promastigotes. Wild type L. donovani promastigotes were processed for immunofluorescence using
either normal rabbit serum (NRS, panel A),
anti-LdPDI (panel B), or anti-BiP (panel C)
antibodies. Bar in panel A represents 10 µm.
View larger version (40K):
[in a new window]
Fig. 5.
Overexpression of LdPDI in L. donovani promastigotes. A, maps of
the endogenous L. donovani LdPDI (WT) and
episomally expressed HA-tagged wild type or mutated LdPDI. The three
proteins contain the same LdPDI putative signal peptide (SP,
black box). The two expressed proteins contain a C-terminal
HA-tag sequence (gray box). Both the wild type (-CGHC-) and
mutated (-AGHA-) active sites are indicated (hatched box).
B, Western blot of lysates of L. donovani
promastigotes transfected with either the expression plasmid alone
(KS) or plasmid encoding LdPDI-wt (PDI-WT) or
LdPDI-mut (PDI-Mut) proteins and reacted with either an
anti-HA, anti-LdPDI, or control rabbit (NRS) antibodies.
Molecular mass in kDa of protein standards is shown on the
left. C, immunofluorescence of transfected
parasites expressing either LdPDI-wt (panels
A-C) or LdPDI-mut (panels
D-F). Promastigotes were reacted
simultaneously with both an anti-HA and an anti-BiP antibody. HA
localization is shown on the red channel in panels
A and D; BiP localization is shown on the
green channel in panels B and E.
Merging of the red and green channels
(yellow) is shown on panel C and F. Bar in panel F represents 10 µm.
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Fig. 6.
Secretion of SAcP by L. donovani
transfectants. A, growth curves of control LdKS
(KS), LdPDI-wt, and LdPDI-mut transfectants. B,
measure of secretory acid phosphatase activity in culture supernatants
of these parasite cultures at the 96-h time point. Acid phosphatase
activity is expressed in nmol/min/107 cells. The difference
in SAcP enzyme activity between LdPDI-mut and LdKS controls was
significant (p = 0.05) by Student's t test
in panel B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. G. Matlashewski for providing the expression plasmid pKS NEO and Dr. J. Bangs for providing the anti-BiP antibody. We also thank Drs. D. Dwyer, R. Duncan, A. Selvapandiyan, and N. Goyal for critical review of this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY166592.
§ Present address: Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Dr. Balmis 148, col Doctores, México D.F., 06726 México.
** To whom correspondence should be addressed: LBPUA, DETTD, OBRR, CBER, Food and Drug Administration, Bldg. 29, Rm. 425, HFM-310, 29 Lincoln Dr., Bethesda, MD, 20892. Tel.: 301-496-1652; Fax: 301-480-7928; E-mail: debrabanta@cber.fda.gov.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M210322200
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ABBREVIATIONS |
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The abbreviations used are: PDI, protein disulfide isomerase; LdPDI, Leishmania donovani PDI (gene in italics, protein in roman); LdPDI-wt, wild type LdPDI; LdPDI-mut, mutant inactive LdPDI; ER, endoplasmic reticulum; SAcP, secretory acid phosphatase; HA, hemagglutinin; PBS, phosphate buffer saline; BPTI, bovine pancreatic trypsin inhibitor; BiP, immunoglobulin heavy chain binding protein.
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