From the Department of Biochemistry and Molecular
Biology and the § Department of Pathology, Oregon Health
Sciences University, Portland, Oregon 97201-3098
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ABSTRACT |
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Hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) is a key enzyme in the purine
salvage pathway of many protozoan parasites. The predicted amino acid
sequences of certain HGPRT proteins from parasites of the
Trypanosomatidae family reveal a COOH-terminal tripeptide signal that
is consistent with the degenerate topogenic signal targeting proteins
to the glycosome, a fuel-metabolizing microbody unique to these
parasites. To determine definitively the intracellular milieu of HGPRT
in these pathogens, polyclonal antiserum to the purified recombinant
HGPRT from Leishmania donovani was generated in rabbits,
and confocal and immunoelectron microscopy were employed to establish
that the L. donovani HGPRT is localized exclusively to the
glycosome. No HGPRT protein was detected in hgprt null
mutants in which both alleles of the HGPRT locus had been
replaced by a drug-resistance cassette. Transfectants of the
hgprt knockout strain in which a wild-type
HGPRT was amplified on an expression plasmid contained
augmented amounts of HGPRT, all of which was localized to the
glycosome.
hgprt transfectants containing amplified
copies of a mutated HGPRT construct in which the Ser-Lys-Val
COOH-terminal targeting signal had been deleted expressed HGPRT
throughout the parasite, including subcellular organelles such as the
nucleus and flagellum. These data demonstrate that the L. donovani HGPRT is compartmentalized exclusively within the
glycosome and that the COOH-terminal tripeptide of the protein is
necessary to achieve targeting to this organelle.
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INTRODUCTION |
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The genus Leishmania, a member of the Trypanosomatidae family, is the causative agent of leishmaniasis, a parasitic disease of considerable import in terms of both mortality and morbidity. All Leishmania sp. are digenetic, existing as extracellular, flagellated promastigotes within their sandfly vectors and as intracellular, nonmotile amastigotes within the phagolysosomes of macrophages and other reticuloendothelial cells of the vertebrate host. The drugs used in the treatment of leishmaniasis and other parasitic diseases have been realized empirically and are far from ideal. Toxicity is common, the drugs are potentially mutagenic and/or carcinogenic, regimens are prolonged and usually require multiple drug administrations, drug resistance or refractoriness persists as a significant therapeutic obstacle, and the prospects for effective antiparasitic vaccines in the immediate future are bleak (1).
The rational development of new antiparasitic drugs that are selective for the metabolic machinery of the parasite will require therapeutic exploitation of fundamental biochemical differences between the parasite and vertebrate host. Perhaps the most striking metabolic discrepancy between parasites and their human hosts is the pathways by which they synthesize purine nucleotides. Whereas mammalian cells synthesize purine nucleotides from amino acids and one-carbon moieties, all protozoan parasites evaluated to date are incapable of purine nucleotide synthesis de novo (2). Thus, these pathogens are auxotrophic for purines and consequently express purine salvage enzymes that enable them to access host purines.
Metabolic, biochemical, and genetic studies have revealed that
Leishmania donovani promastigotes funnel a variety of
exogenous purines into hypoxanthine (3, 4), intimating that the enzyme hypoxanthine-guanine phosphoribosyltransferase
(HGPRT)1 plays a central role
in this purine acquisition process. However, the viability and normal
growth rate of hgprt null mutants of L. donovani that have been created by targeted gene replacement have
clearly demonstrated that HGPRT does not play an indispensable role in
purine salvage by the promastigote form of the parasite (5, 6). Whether
a functional HGPRT activity is crucial to the infective form of the
parasite is unknown. The trypanosomatid HGPRT also initiates the
intracellular metabolism of allopurinol (HPP, 4-hydroxypyrazolo[3,
4]pyrimidine), an effective antileishmanial and antitrypanosomal agent
(7, 8) that is nontoxic to humans and widely used in the treatment of
hyperuricemia and gout (9). Indeed, HPP has demonstrated significant
therapeutic efficacy in patients with either cutaneous leishmaniasis
(10) or chronic Chagas disease (11).
Subcellular fractionation experiments have suggested that the HGPRT
activities in Leishmania mexicana (12), Trypanosoma brucei (13), and Trypanosoma cruzi (14) are localized
to the glycosome, a fuel metabolizing microbody that is unique to
parasites of the Trypanosomatidae family (15, 16). A glycosomal milieu for HGPRT in the Trypanosomatidae is supported by the fact that the
COOH-terminal tripeptides of the HGPRTs from L. donovani
(17), T. cruzi (18), and Crithidia fasciculata
(19) are consistent with the acceptable degeneracy for the well
characterized COOH-terminal targeting signal for glycosomal
localization (20, 21). As definitive information on the subcellular
location of therapeutically pertinent enzymes is critical for drug
targeting, we have established by confocal and immunoelectron
microscopy that the HGPRT protein from L. donovani, unlike
the mammalian HGPRT, is localized exclusively to the glycosome.
Furthermore, we have demonstrated genetically that the COOH-terminal
tripeptide is required for this glycosomal compartmentation, as
hgprt mutants transfected with an hgprt construct truncated at the COOH terminus express HGPRT protein throughout the parasite.
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EXPERIMENTAL PROCEDURES |
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Parasites-- L. donovani promastigotes were grown at room temperature in Dulbecco's modified Eagle's-Leishmania medium with xanthine as the purine source (22). Cells employed in the biochemical or immunolocalization experiments were routinely harvested in exponential growth at a density of ~5-6 × 106 cells/ml. The wild-type DI700 strain originates from the 1S Sudanese clone of L. donovani and is designated DI700:H+/+ for the purposes of the genetic manipulations described in these studies, where H denotes the HGPRT locus and + specifies the wild-type allele. Mutant parasites containing functional copies of the neomycin or hygromycin phosphotransferase genes were routinely maintained in Dulbecco's modified Eagle's-Leishmania medium supplemented with 50 µg/ml Geneticin® (G418) (Life Technologies, Inc.) or 400 µg/ml hygromycin, respectively.
Construction of Leishmanial Expression Vectors and Transfection
into L. donovani--
The L. donovani HGPRT and flanking
regions were isolated as described (5, 17). Vectors pX63-HYG
and pSNBR (containing the neomycin phosphotransferase gene) have been
described (23, 24) and were provided by Dr. Stephen M. Beverley of
Washington University. The construction of the hgprt null
mutant, DI700:Hn/n has also been described previously (5),
where the n superscript designates an allele in which the
neomycin phosphotransferase marker has been inserted into the
HGPRT locus. The DI700:Hn/n strain was generated by
replacing the first wild-type HGPRT allele with a targeting
construct encompassing the neomycin phosphotransferase marker followed
by negative selection for loss of heterozygosity to generate the
hgprt knockout (5). The DI700:Hn/n strain was
employed as a recipient for transfection of functional episomal
HGPRT constructs.
Expression of Wild-type and Mutant HGPRT Constructs in E. coli--
The construction of the wild-type HGPRT pBAce
expression vector (17) and the wild-type HGPRT-pSelect mutagenesis
expression vector (24) have been reported. To generate an
HGPRT expression vector in which the COOH-terminal
tripeptide was deleted, the HGPRT within pSelect (Promega,
Madison, WI) was altered by introduction of three stop codons (boldface
in primer below) at positions 209-211 of the protein. The mutagenic
primer was
5-CTCAAGAAGGAGTACTACGAGAAGCCGGAGTGATAGTAGTAGCGGTGACGAGCTATACTCGAG-3
and contained a PstI site (underlined) at the 3
end.
The mutated NdeI-PstI HGPRT fragment
from pSelect was then ligated into the appropriate sites in pBAce and
transformed into S
606 (
pro-gpt-lac, thi, hpt) E. coli, a strain lacking the bacterial hypoxanthine and
xanthine-guanine phosphoribosyltransferase enzymes (26). The
COOH-terminal truncation was verified by nucleotide sequencing.
Enzyme Purification and Assay-- Recombinant wild-type and mutant HGPRT were purified to homogeneity by affinity chromatography as reported (17, 27). Activity of recombinant enzyme was assayed spectrophotometrically as described (25). HGPRT activity in sonicated extracts of exponentially growing parasites (~1.0 × 107 parasites/ml) that had been extensively washed with phosphate-buffered saline (PBS) was assessed by a previously described radiometric protocol (27). Protein was measured by the method of Bradford (28).
Antibodies-- Polyclonal antiserum to recombinant HGPRT was generated as described (5). The antisera was purified by chromatography over a protein A-Sepharose column (Sigma) according to the brochure of the supplier. Rabbit antiserum raised to the glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein of L. mexicana were generously provided by Dr. P. Michels and F. Opperdoes of the International Institute of Cellular and Molecular Pathology in Brussels, Belgium.
Immunoblotting-- Wild-type, mutant, and transfectant parasites were harvested by centrifugation, washed with PBS, resuspended in sample buffer, and heated to 100 °C for 5 min. Lysates from 105 cells in a volume of 20 µl were fractionated on a 15% SDS-polyacrylamide gel (29) and electrophoretically transferred to a nitrocellulose membrane as described (30). The nitrocellulose filters were incubated with anti-HGPRT antisera at a 1:10,000 dilution using a blocking solution of 5% milk, and the blots were developed using the ECL chemiluminescence system (Amersham Corp.) and goat anti-rabbit IgG coupled to horseradish peroxidase (Boehringer Mannheim).
Immunofluorescence Microscopy--
Exponentially growing
L. donovani at concentrations ~5-6 × 106 cells/ml were harvested by centrifugation, washed
several times in PBS, incubated for 30 min in fixative consisting of 50 mM Hepes buffer, pH 7.2, 4% paraformaldehyde, and 0.1%
glutaraldehyde, and attached to coverslips coated with
poly-L-lysine. Cells were then permeabilized for 30 min at
ambient temperature in PBS containing 1% Triton X-100 and 5% goat
serum. Anti-HGPRT antibody at a dilution of 1:200 was added to the
permeabilized cells and incubated either for 4 h at 25 °C or
for 16 h at 4 °C. Coverslips were rinsed six times with PBS,
incubated with goat anti-rabbit IgG secondary antibody conjugated to
Texas Red fluorophore (Molecular Bioprobes, Eugene, OR) at a 1:500
dilution, and incubated overnight at 4 °C in the absence of light.
After six more washes in PBS, a fluorescein lipophilic dye,
3,3-diohexyloxocarbocyanine iodide (Molecular Bioprobes), was added to
a concentration of 1 µg/ml, and the coverslips were washed
immediately with PBS. Coverslips were mounted on slides in 50 mM Tris, pH 8.0, 90% glycerol, and 20 mg/ml
n-propylgallate and examined with a confocal laser scanning
microscope as described (31).
Immunoelectron Microscopy-- Cells were harvested and fixed either with 8% paraformaldehyde in 50 mM Hepes buffer, pH 7.2, or as described above for the immunofluorescence microscopy. After fixation, cells were washed three times with 100 mM Hepes buffer, pH 7.2, then infused with polyvinylpyrrolidone and sucrose (32) for ultrathin cryosectioning. Double labeling using two different sizes of gold probes (Amersham Corp.) was performed as described (33). 1% glutaraldehyde in PBS was used for the blocking step between antibodies. Control incubations included labeling cryosections with either 1) anti-HGPRT antibodies competed with purified recombinant HGPRT, 2) no primary antibodies, or 3) irrelevant antibodies. Immunolabeled sections were embedded in 25 centipoise methyl cellulose and uranyl acetate (34) and viewed with a Philips 301 transmission electron microscope (Philips Electronics, Mahwah, NJ).
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RESULTS |
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Antibody Characterization--
To determine the location of the
HGPRT in L. donovani, antibodies directed against epitopes
on the enzyme were generated in rabbits. These antibodies recognized
only a single protein in wild-type parasite lysates that migrated with
a molecular mass approximate to that predicted from the translated
HGPRT protein coding region (Fig.
1). This band was established to be HGPRT genetically as this antiserum did not recognize any protein in lysates
of the hgprt DI700:Hn/n strain (Fig. 1).
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HGPRT Is Targeted to L. donovani Glycosomes-- The location of the HGPRT enzyme in wild type L. donovani was initially assessed by confocal microscopy. Immunofluorescence analysis of HGPRT stained with Texas Red-conjugated secondary antibody revealed a punctate pattern in DI700:H+/+ cells not observed with DI700:Hn/n parasites (Fig. 2, A and B, respectively). Immunoelectron microscopy was used to confirm glycosomal targeting of the enzyme (Fig. 3). Gold particle labeling for HGPRT was sequestered within the glycosomes in wild-type parasites, and no other subcellular compartments were labeled to a significant degree (Fig. 3, A and B). This localization pattern paralleled that obtained with antibodies to L. mexicana GAPDH (Fig. 3C), a glycolytic enzyme whose glycosomal location in L. donovani has been documented (35, 36). Furthermore, the L. donovani HGPRT colocalized with the GAPDH (Fig. 3D). No immunogold labeling was observed for either DI700:H+/+ cells incubated with excess recombinant HGPRT (Fig. 3E) or for DI700:Hn/n parasites (Fig. 4, A and B). These data demonstrate clearly that the L. donovani HGPRT is compartmentalized to the glycosome.
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The COOH-terminal Ser-Lys-Val Tripeptide Is Necessary for
Glycosomal Targeting--
To validate whether the COOH-terminal
tripeptide is required for targeting HGPRT to the glycosome,
DI700:Hn/n parasites were transformed with expression plasmids
containing either a wild-type copy of HGPRT (pX63-HYG-HGPRT)
or a mutated construct lacking the codons for the SKV COOH-terminal
tripeptide (pSNBR-HYG-hgprt(209-211)). Both the
DI700:Hn/n[pX63-HYG-HGPRT] and
DI700:Hn/n[pSNBR-HYG-hgprt(
209-211)]
transfectants expressed ~10-15-fold greater quantities of HGPRT
activity (Fig. 5) and protein (Fig. 1)
than DI700:H+/+ parasites (Fig. 5). Deletion of the
Ser-Lys-Val tripeptide did not appear to grossly alter the catalytic
properties of the enzyme. Apparent Km and
kcat values calculated for the mutant enzyme by
Hanes analysis were 4.8 µM and 6.2 s
1 for
hypoxanthine and 11.6 µM and 15.8 s
1 for
guanine. These values are comparable with the Km and
kcat values of 6.4 µM and 5.7 s
1 for hypoxanthine and 9.9 µM and 12.1 s
1 for guanine that were reported previously for the
wild-type L. donovani HGPRT (17).
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DISCUSSION |
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The HGPRT enzyme of L. donovani has been definitively
localized to the parasite glycosome by indirect immunofluorescence and immunoelectron microscopy. Specificity of antibodies directed against
HGPRT epitopes used for the immunocytochemistry was established by 1)
immunoblotting of whole cell lysates, 2) the ability of excess purified
recombinant enzyme to block immunogold labeling of the parasites, and
3) the failure of the anti-HGPRT antisera to recognize any protein in
hgprt parasites. A glycosomal milieu for this central
enzyme in purine salvage in trypanosomatids was initially conjectured
on the basis of crude subcellular fractionation experiments in which
HGPRT activity was largely associated with the glycosomal fraction of
L. mexicana (12), T. brucei (13), and T. cruzi (14). Moreover, trypanosomatid HGPRT coding
sequences predicted COOH-terminal tripeptides compatible with the well
established COOH-terminal signal that is recognized by the glycosome
import machinery (20, 21). Of these three amino acids, the first is
usually a small uncharged amino acid, the second an amino acid containing a hydrogen bond-forming group, and the third is hydrophobic in nature (20, 21). The L. donovani, T. cruzi,
and C. fasciculata HGPRTs terminate in Ser-Lys-Val (17),
Ser-Lys-Tyr (18), and Ser-Lys-Leu (19), respectively, and are all
within the acceptable degeneracy for this targeting signal. The
T. brucei HGPRT ends with an Ala-Lys-Arg (37) and would not
be predicted from mutational studies of targeting signals on firefly
luciferase (21) and chloroamphenicol acetyltransferase-phosphoglycerate
kinase hybrids (20) to be a glycosomal import signal.
Mutational analysis has established that the Ser-Lys-Val of the
L. donovani HGPRT protein is required for glycosome
compartmentalization. Transfection of the
pSNBR-HYG-hgprt(209-211) construct in which these three
amino acids have been deleted into a
hgprt background results in a ubiquitous distribution of the protein throughout the
parasite, including the nucleus, flagellum, and plasma membrane, as
well as glycosomes. This mislocalization of mutated protein cannot be
attributed to plasmid expression of HGPRT, as a wild-type episomal construct directed the expression of correctly targeted protein, or to major structural abnormalities, as the
hgprt(
209-211) protein was kinetically comparable with the
wild-type enzyme. Although, the COOH-terminal tripeptide is necessary
for glycosome compartmentation, and whether it contains all the
necessary information for targeting HGPRT to the glycosome is unknown.
However, Ser-Lys-Val addition to the COOH terminus does mediate
glycosome entry of firefly luciferase in T. brucei (21).
The reason for the glycosomal compartmentalization of HGPRT in L. donovani and related parasites remains unclear, as the enzyme is cytosolic in mammalian cell systems. However, glycosomes accommodate a variety of fuel metabolizing enzymes that perform essential nutritional functions for the parasite (15, 16), and the auxotrophy of these parasites for purines (2) dictates that purine salvage is nutritionally essential for these organisms. Therefore, the perspective that glycosomes are fuel metabolizing organelles can perhaps be expanded to include other nutritional pathways. The location of other purine salvage and interconversion enzymes in trypanosomatids has not been conclusively determined. Fractionation of L. mexicana extracts by isopycnic centrifugation indicated that the purine salvage enzymes, IMP dehydrogenase, the enzyme that converts the IMP product of HGPRT to XMP, and xanthine phosphoribosyltransferase, cosedimented with glycosome and particulate fractions, respectively, whereas adenine phosphoribosyltransferase, nucleosidases, and adenine deaminase were all cytosolic (38). Interestingly, the COOH-terminal tripeptides of IMP dehydrogenase (39) and xanthine phosphoribosyltransferase (Ala-Lys-Leu, data not reported) from L. donovani are both plausible glycosomal import signals. The comparable sequence on adenine phosphoribosylransferase, His-Pro-His, is an unlikely candidate to mediate glysosome entry (40). One credible hypothesis for the glycosomal compartmentation of HGPRT is to provide optimal access to substrate. However, purine bases must obligatorily be acquired from the extracellular environment, whereas PRPP substrate is generated from ribose-5-phosphate and ATP by the catalytic action of PRPP synthetase. Although the location of the parasite PRPP synthetase is not known, the enzyme terminates in an Arg-Asp-Ser sequence, a dubious targeting signal for glycosome entry. Despite the lack of a clear rationale for the atypical organellar compartmentalization of HGPRT in trypanosomatids, the categorical association of this therapeutically germane purine salvage enzyme to the glycosome is significant not only from a biological perspective but also from a drug development standpoint, as drugs that target HGPRT may need to traverse intracellular membranes to exert their antiparasitic effects.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. Michel and F. Opperdoes for providing antibodies to the L. mexicana GAPDH protein, K. Fish for advice and expertise in confocal microscopy, and E. Snapp for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by Grant AI-23682 from the NIAID, National Institutes of Health.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.
¶ Burroughs Wellcome Fund Scholar in Molecular Parasitology, supported in part by a grant from the Burroughs Wellcome Fund, and to whom correspondence should be addressed. Tel.: 503-494-8437; Fax: 503-494-8393; E-mail: ullmanb{at}ohsu.edu.
1 The abbreviations used are: HGPRT, hypoxanthine-guanine phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase gene; APRT, adenine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPP, 4-hydroxypyrazolo[3,4]pyrimidine, allopurinol; HYG, hygromycin phosphotransferase gene; NEO, neomycin phosphotransferase gene; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PRPP, phosphoribosylpyrophosphate; bp, base pair.
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REFERENCES |
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