From the Institute for Cell Biology, University of Bern, Baltzerstrasse 4, Berne CH-3012, Switzerland
Received for publication, June 21, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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This study reports the identification and
characterization of a cAMP-specific phosphodiesterase from the
parasitic hemoflagellate Trypanosoma brucei. TbPDE2A is a
class I phosphodiesterase. Its catalytic domain exhibits 30-40%
sequence identity with those of all 11 mammalian phosphodiesterase
(PDE) families, as well as with PDE2 from
Saccharomyces cerevisiae, dunce from
Drosophila melanogaster, and regA from
Dictyostelium discoideum. The overall structure of TbPDE2A
resembles that of human PDE11A in that its N-terminal region contains a
single GAF domain. This domain is very similar to those of the
mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential
cGMP binding site. TbPDE2A can be expressed in S. cerevisiae, and it complements an S. cerevisiae PDE
deletion strain. Recombinant TbPDE2A is specific for cAMP, with a
Km of ~2 µM. It is entirely
resistant to the nonselective PDE inhibitor
3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin,
dipyridamole, sildenafil, and ethaverine with IC50 values
of 5.4, 5.9, 9.4, and 14.2 µM, respectively. All four
compounds inhibit proliferation of bloodstream form trypanosomes in
culture, indicating that TbPDE2A is an essential enzyme.
Cyclic nucleotide-specific phosphodiesterases represent a large
and divergent group of enzymes. In eukaryotes, two classes of
phosphodiesterases (PDEs)1
have been recognized (1, 2). Class I includes all currently known
families of mammalian PDEs (see below), as well as a number of PDEs
from lower eukaryotes, such as PDE2 from the yeast Saccharomyces cerevisiae or the product of the regA gene of
Dictyostelium discoideum. All class I enzymes show a
considerable extent of amino acid sequence conservation within their
catalytic domains. In contrast, the class II PDEs show little sequence
similarity to the class I enzymes, and their Km are
generally much higher than those of the class I enzymes. Class II PDEs
were identified (e.g. in S. cerevisiae (1),
D. discoideum (3), and Candida albicans (4)).
Class I PDEs have been studied particularly well in mammals, where 11 distinct PDE families have been identified to date, based on their
substrate specificities, kinetic properties, allosteric regulators,
inhibitor sensitivities, and amino acid sequences. Each family exhibits
>50% amino acid sequence identity within the conserved catalytic
domain of about 250 amino acids length, while sequence identity between
different families is only 30-40% in the same region (5). Family
members also share extensive similarity in regions outside the
catalytic domain, while no significant similarity of that region can be
detected between different families. Besides their amino acid
sequences, the different families also display distinctive
pharmacological properties, which form the basis for the development of
family-specific PDE inhibitors for clinical use (5-9). Genetic defects
in PDE genes have been suspected as the underlying causes of a number
of diseases (10-13), although the relationship between PDE genes and
disease is clearly established only between PDE6 and some forms of
retinal degenerative disease (11).
PDEs have become highly attractive targets for drug development over
the last few years. A growing number of family-specific and
subtype-specific inhibitors have been developed despite the considerable sequence conservation between the catalytic domains of
different families. Several PDE inhibitors are being used or are under
exploration for ailments as diverse as autoimmune disease (14, 15),
arthritis (16), asthma (17, 18), and impotency (19, 20) and as
anti-inflammatory agents (21). The ongoing development of new and ever
more subtype-specific inhibitors holds great promise for achieving more
specific drug action with fewer side effects as well as new areas for
the application of PDE inhibitors. In view of the potential of PDE
inhibitors as chemotherapeutics, it is surprising how little is
currently known about PDEs of parasites as possible drug targets.
The African trypanosome Trypanosoma brucei is a eukaryotic
microorganism that causes the fatal human sleeping sickness (22), as
well as Nagana, a devastating disease of domestic animals in large
parts of sub-Saharan Africa. Chemotherapy of human trypanosomiasis is
in a dismal state (23). New drugs and drug targets are urgently required, and the cyclic nucleotide-specific PDEs may constitute a
class of new drug targets. cAMP signaling in trypanosomes is still
largely unexplored (24, 25). cAMP is involved in the regulation of
differentiation of bloodstream form trypanosomes from the proliferative
"long slender" forms to the insect-preadapted, nonproliferative
"short stumpy" forms (26). Several gene families for adenylyl
cyclases have been identified in T. brucei (24, 27,
28).2 Even less is known
about trypanosomal phosphodiesterases. An early study demonstrated PDE
activity in cell lysates of bloodstream form T. brucei (29),
and experiments with PDE inhibitors suggested that interference with
PDE activity might affect cell differentiation (26, 30). A recent
report (31) described a PDE activity in cultured Leishmania
mexicana. This enzyme is partly soluble and partly particulate,
and its characteristics indicate that it may represent a class II
PDE.
The current study describes the identification and characterization of
a cAMP-specific PDE from T. brucei (TbPDE2A) with
considerable amino acid sequence similarity to the class I PDEs. This
classification is further supported by its low Km
for cAMP as a substrate. In its N terminus, TbPDE2A contains a single
GAF domain (32), which may represent a noncatalytic cGMP-binding site.
TbPDE2A is a representative of a small, dispersed
family of similar, but nonidentical genes. It can be expressed in the
yeast S. cerevisiae, and it complements the heat-shock
susceptibility phenotype of a PDE1/PDE2 deletion strain.
Inhibitor studies showed that TbPDE2A is fully resistant to most
inhibitors tested, including broad spectrum inhibitors such as IBMX.
However, the enzyme is sensitive to the family 5 inhibitors
dipyridamole and sildenafil, the family 3 inhibitor trequinsin, and the
calcium-channel blocker ethaverine.
Materials--
Radiochemicals were purchased from PerkinElmer
Life Sciences. Barium hydroxide solution was purchased from
Sigma. PDE inhibitors were from the following sources: IBMX, zaprinast,
ethaverine, papaverine, theophylline, milrinone, trequinsin,
vinpocetine, 8- methoxymethyl-IBMX, dipyridamole, and rolipram were
from Sigma; erythro-9-(2-Hydroxy-3-nonyl) adenine, HCl,
zardaverine, cilostamide, and Ro-20-1724 were from BioMol (Plymouth
Meeting, PA); etazolate and pentoxiphylline were from Calbiochem; and
sildenafil citrate was a gift from Pfizer Central Research (Sandwich,
Kent, UK). DNA sequencing was outsourced to Microsynth GmbH (Balgach,
Switzerland). Reactions were run with BigDye terminators
(PE-Biosystems) and were analyzed on an ABI Prism 377 instrument.
Cell Culture--
Trypanosoma brucei strain 427 (derived form
MiTat 15a) was grown as procyclic form at 27 °C in SDM medium (33).
Monomorphic bloodstream forms of strain 221 (MiTat 1.2) were cultivated
as described by Hesse et al. (34). The yeast strain PP5
(MATa leu2-3 leu2-112 ura3-52 his3-532 his4 cam
pde1::URA3 pde2::HIS3; Ref. 35) was a gift of John
Colicelli (UCLA). Yeast transformation was done as described (36).
Transformants were selected on synthetic minimal medium containing
0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose,
supplemented with an amino acid mixture lacking leucine (SC-leu). Heat
shock experiments were performed by replica-plating patches onto YPD
plates prewarmed to 55 °C, and the heat shock was continued for 15 min. After cooling the plates to room temperature, they were incubated
for 2-3 days at 30 °C.
TbPDE2A Constructs--
Screening of the T. brucei
EST data base for class I PDE homologs identified plasmid pT2928
(accession number W84103) as coding for a potential T. brucei PDE. The cDNA fragment of pT2928 was completely
sequenced and shown to represent the catalytic domain and the complete
3'-untranslated region of a candidate phosphodiesterase. This gene was
termed TbPDE2A. The cDNA of pT2928 was then used
to screen a genomic library of T. brucei in
The 5'-end of the open reading frame was amplified from a fragment of
genomic DNA, using the forward primer pde2gf2
(5'-GAGAATTCAAACATGTATGTGCACGACGTACGCATGTTC-3'), containing an EcoRI site (underlined) followed by a Kozak
sequence and the start codon (boldface type, underlined), and the
reverse primer pde2gr (5'-TTCAACCCCATATGATCAAGATCATGCACCAG-3'). The PCR product (804 bp) was cloned into pGEM-T-Easy, verified by sequencing, and then excised by digestion with EcoRI and NdeI
and cloned into pTPDE23U cut with the same enzymes. This step generated
a full-length copy of TbPDE2A (pTPDE2A) containing an NcoI
site immediately before the stop codon. The aim of these manipulations
was to obtain a TbPDE2A gene with an unaltered open
reading frame but with restriction sites introduced upstream
(EcoRI) and downstream (XhoI, BamHI) of the start and stop codons, respectively, to facilitate further cloning steps. In addition, a Kozak sequence for efficient expression in S. cerevisiae was introduced in front of the start codon,
as well as an NcoI site immediately before the stop codon
for the later insertion of immunological tags (see below).
For generating an N-terminally truncated form of TbPDE2A without the
noncatalytic cGMP-binding domain (starting at Met124
of the full sequence), the corresponding region was amplified from
genomic DNA using the forward primer pde2gf1
(5'-GAGAATTCAAACATGGAAGTTAACGAACACCGAGCAACATTG-3'), containing an EcoRI site (underlined) followed by a Kozak
sequence and the codon for Met124 (boldface type,
underlined), and the reverse primer pde2gr (see above). The PCR product
(475 bp) was cloned and sequenced as indicated above. Finally, it was
excised by digestion with EcoRI and NdeI and
inserted into the corresponding sites of pTPDE23U to generate pTPDE2AT.
The various constructs are outlined in Fig.
1.
For inserting a hemagglutinin tag (amino acid sequence: YPYDVPDYAGIPM)
at the C terminus of both constructs, two complementary oligonucleotides, Htfor
(5'-CATGGTTACCCATACGATGTCCCAGATTACGCCGGTATTCCAATGTAGG-3'; open NcoI site underlined, stop signal in boldface type and
underlined) and Htrev
(5'-GATCCCTACATTGGAATACCGGCGTAATCTGGGACATCGTATGGGTAAC-3'; open BamHI site underlined), were annealed and then inserted
into pTPDE2A and pTPDE2AT digested with NcoI and
BamHI. The resulting tagged constructs (pTPDE2Ahm and
pTPDE2AThm) were finally verified by sequencing. Similar constructs
were also made that contain the TY-1 tag (38) instead of the
hemagglutinin tag at their C termini.
For expression in S. cerevisiae, the hemagglutinin-tagged
genes were introduced into the yeast expression vector p425CYC1 containing an attenuated CYC1 promotor (39) and into pLT1, which allows
high level expression from a strong TEF2 promotor. pLT1 was derived
from p425CYC1 by replacing its expression cassette with the TEF2
promotor, including the original TEF2 Kozak sequence. The initiation
codon is followed by two restriction sites, which allow cloning of the
gene to be expressed. The resulting sequence of the expression site is
as follows: TEF2 promotor ( Yeast Cell Lysis--
Yeast cells grown to midlog to end log
phase in SC-leu medium were collected, resuspended quickly in the
original volume of prewarmed YPD medium, and incubated for an
additional 3 h at 30 °C to maximize protein expression. Cells
were then harvested and washed once in H2O and once in HBB
buffer (Hank's balanced salt solution, containing 50 mM
HEPES, pH 7.5). The washed cell pellet was suspended in an equal volume
of HBB containing a protease inhibitor mixture (CompleteTM; Roche
Molecular Biochemicals). Cells were lysed by grinding with glass beads
(425-600 µm; Sigma) in 2-ml Sarstedt tubes using a FastPrep FP120
(3 × 45 s at setting 4). After cell breakage, a hole was
punched in the bottom of the tube with a needle, the tube was placed on
top of a 5-ml plastic tube, and it was centrifuged in an SS34 rotor for
6 min at 6000 rpm. This step left the glass beads in the Sarstedt tube,
while the cell lysate was collected in the plastic tube, where unbroken cells and large cell fragments formed a pellet. The supernatant was
transferred to a fresh tube and was clarified by centrifugation for 15 min at 15,000 × g. To the resulting supernatant,
glycerol was added to a final concentration of 25% (v/v), and it was
stored at Phosphodiesterase Assay--
PDE assays were done using the
ZnSO4/Ba(OH)2 precipitation method of Schilling
et al. (40). This assay was chosen because it can
potentially be upgraded to high throughput screening and because it
produces less liquid radioactive waste than other procedures. The assay
was validated and found to provide results essentially identical to
those with other assay formats (40). The reaction contained 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 10 mM MgCl2, and [3H]cAMP or
[3H]cGMP (50,000 dpm/reaction) in a total volume of 100 µl. Incubation was at 30 °C for 20 min. Reactions were stopped by
the addition of 50 µl of 21.5 mM ZnCl2,
followed by 50 µl of 9 mM Ba(OH)2 and incubated on ice for 30 min. The precipitates were filtered through GF-C glass fiber filters, and filters were washed three times with 1 mM NaOH, 100 mM NaCl. The filters were dried
and counted in liquid scintillation fluid (4 g/liter omnifluor in
toluene). All assays were carried out in triplicates and with three
independent enzyme preparations. Controls for the efficiency of
precipitation of cAMP and of AMP were always included. AMP was
precipitated with an efficiency in the range of 55%, while the
contamination of the precipitate with cAMP was in the range of
0.6-0.8% of the input. The assay worked reliably over all cAMP
concentrations used, up to 50 µM. When assaying yeast
cell extracts, control lysates from the PDE deletion strain transfected
with empty vector were used as background controls. These control
lysates never exhibited detectable activity beyond the background
produced without lysate. The efficiency of 5'-AMP precipitation
remained unchanged when yeast lysates were incubated for up to 2 h
under assay conditions, indicating that the assay is not affected by a
potential conversion of 5'-AMP to adenosine or adenine. Inhibitor
studies were done at a cAMP concentration of 1 µM,
i.e. close to the Km of TbPDE2A, so that
the IC50 values should approximate the
Ki. Inhibitors were dissolved in Me2SO
or ethanol, and the final concentration of the solvent never exceeded
1% in the assay reaction. Incubation times and enzyme concentrations
were always adjusted so that less than 30% of the input substrate was
hydrolyzed (2-5 µg of total protein/100-µl assay).
IC50 values were calculated by curve fitting on a
four-parameter dose-response model with variable slope, using the Prism
software package of Graph Pad Inc. (San Diego, CA).
Cytotoxicity Determination--
Cytotoxicity of PDE inhibitors
was determined for bloodstream forms in culture by determining acid
phosphatase activity as described (41). Exponentially growing
monomorphic bloodstream forms MiTat 1.2 were transferred into
colorless medium (42) (cell density 3 × 105 cells/ml
culture) and were seeded into microtiter wells (199 µl/well)
containing 1 µl of inhibitor or solvent control. Plates were
incubated for both 20 and 40 h at 37 °C in a humidified
incubator with a 5% CO2 atmosphere. At the end of the
growth period, cells were lysed by the addition of 20 µl of
lysis/substrate buffer (20 mg/ml p-nitrophenylphosphate in 1 M sodium acetate, pH 5.5, 1% Triton X-100), and the
incubation was continued for another 4 h at 37 °C. Production
of p-nitrophenol was determined at 405 nm on a microtiter
plate reader. To control for intrinsic absorbance by the inhibitors,
control series containing inhibitor dilutions but no cells were run for
every experiment, and the resulting absorbance values were subtracted
as background from the experimental readings. All assays were run in triplicates.
cAMP Determination--
Intracellular cAMP concentrations were
determined using an enzyme-linked immunoassay kit (BioMol). Bloodstream
form trypanosomes (5 × 106/ml) were incubated with
inhibitors at the concentrations and for the times specified. At the
end of the incubation, 1-ml aliquots were centrifuged at 3,000 rpm for
8 min to collect the cells. The cell pellet was suspended in 150 µl
of 0.1 N ice-cold HCl. After incubation in ice for 20 min,
the suspension was centrifuged at 12,000 rpm for 10 min. The HCl
precipitate was processed for protein determination, while 100 µl of
the cleared supernatant was transferred to a fresh tube and used to
assay the cAMP content, following the instructions of the supplier of
the kit. Assays were performed in triplicates, and two independent
experiments using different batches of cells were performed. The cAMP
content of control cells as determined with this procedure was 66 ± 14 pmol/109 trypanosomes, corresponding well with values
obtained with different trypanosome strains and a different cAMP assay
(26).
The TbPDE2A Locus--
Upon searching the T. brucei
expressed sequence tag data base for potential phosphodiesterase genes,
an expressed sequence tag clone (pT2928) was identified. The
corresponding plasmid was obtained (courtesy Philip Majiwa, ILRI) and
sequenced. The cDNA fragment contained the 3'-part of a cDNA
that unambiguously represented a phosphodiesterase gene, termed
TbPDE2A according to the recently proposed rules for the
nomenclature of trypanosomatid genes (43). Southern blot analysis of
genomic DNA demonstrated that TbPDE2A is not a single gene
but rather a member of a small gene family (Fig.
2A). This was further
confirmed by screening a procyclic cDNA
library,3 which resulted in
the identification of several cDNA clones that represent different
PDE2 family members (unpublished results). The cDNA fragment from
pT2928 was then used to screen a genomic library of T. brucei, and the TbPDE2A locus was recovered on a 6-kilobase pair genomic EcoRI DNA fragment. The fragment was
sequenced, as were several cDNA clones for TbPDE2A. The
organization of the TbPDE2A locus (Fig. 2B)
demonstrates that it contains three different, closely spaced genes.
The first one is a RIME element (nucleotides 376-876), a member of a
family of abundant, highly transcribed, repetitive transposable
elements (44). Within this element, nucleotides 868-632 on the reverse
strand represent the open reading frame coding for a RIME-associated
protein. The RIME element is flanked by two 12-bp direct repeats
(nucleotides 364-375 and 877-888). The open reading frame for TbPDE2A
extends from nucleotide 1770 to 3225 and codes for a protein of 485 amino acids. The predicted initiator methionine codon was functional,
and the predicted open reading frame coded for an active protein when
expressed in S. cerevisiae (see below). The coding region is
followed by a long 3'-untranslated region of 1196 nucleotides, and the
poly(A)-addition site is represented by nucleotide 4420. Downstream of
the TbPDE2A gene, a gene for a member of the NHP2/RS6 family
of nuclear proteins (45) is coded for by nucleotides 4635-5062. This
is the first instance where such a gene was detected in T. brucei, and the role of the corresponding protein remains unknown.
The presence of unrelated genes upstream and downstream of
TbPDE2A demonstrated that the members of this PDE
family are not closely linked.
Expression of TbPDE2A was analyzed both by Northern blot hybridization
and by reverse transcriptase-polymerase chain reaction. Both approaches
demonstrated that TbPDE2A is expressed both in the bloodstream and the
procyclic (insect stage) form of the trypanosome life cycle (data not shown).
Predicted Amino Acid Sequence of TbPDE2A--
The open reading
frame of TbPDE2A predicts a protein of 485 amino acids, with a
calculated molecular mass of 55,348 (Fig. 3). The N terminus of TbPDE2A contains a
single GAF domain (Val3-Val117; Ref.
32), which may function in cGMP binding. The presence of a single GAF
domain in TbPDE2A is reminiscent of the human PDE11A, which also has a
single GAF domain, while all other mammalian PDEs with such domains
(PDE2, -5, -6, and -10) contain two of them in a closely spaced
arrangement. The overall sequence identity between the single GAF
domain of TbPDE2A and either of the corresponding domains of mammalian
PDE2, -5, -6, -10, and -11 varies between 30 and 50%, with several
residues (Leu59, Cys60, Pro62,
Asn77, Lys78, Phe88, and
Asp91) strongly or absolutely conserved. For mammalian
PDE5A, where cGMP-binding by the GAF domain was experimentally
demonstrated, the interaction with cGMP was predicted to occur via
Asn77, Lys78, and Asp91, all of
which are strongly conserved (46).
The catalytic domain of TbPDE2A is located between Phe205
and Phe438, as predicted by analogy with other PDEs. All
class I PDEs contain a conserved region of ~250 amino acids that
represents the catalytic domain (47). Several residues within this
domain are absolutely or chemically conserved between PDE families and
across species from yeast to humans. The predicted catalytic domain
contains the signature sequence for cyclic nucleotide-specific
phosphodiesterases (His269-Tyr281) (48). Two
putative metal-binding motifs are represented by His229,
His233, and Glu252 and by His269,
Asp270, His273, and Glu302,
respectively (49). A recent structural analysis of human PDE4 has
directly demonstrated that the first of the two motifs binds a
Zn2+ atom (50), while the metal ligand for the second motif
remains to be determined. The putative nucleotide-binding site is
formed by amino acids Lys389-Phe438 (51). The
neighboring histidine residues (His304 and
His305), which are located outside this conserved
nucleotide binding region, may correspond to the vicinal histidine
residues shown to be involved in cAMP binding in the human PDE4A (52).
Many amino acid residues of the catalytic domain are highly conserved between TbPDE2A and representatives of the 11 mammalian PDE families (His229 (identical between TbPDE2A and 10 out of the 11 mammalian PDE families), Asn230 (10/11), His269
(11/11), Asp270 (11/11), Asp272 (10/11),
His273 (11/11), Gly275 (11/11),
Asn278 (10/11), Glu302 (11/11),
His304 (11/11), His305 (11/11),
Ala342 (11/11), Thr343 (11/11),
Asp344 (11/11), Asp383 (11/11),
Glu404 (11/11), Phe405 (9/11),
Gln408 (10/11), Gly409 (9/11),
Asp410 (11/11), Asp424 (9/11),
Gln435 (11/11), and Phe438 (10/11)).
Interestingly, the linker region between the cGMP-binding domain and
the catalytic domain contains a phosphorylation site for cAMP/cGMP
kinases (Lys144-Thr147). The functional
significance of this regulatory site remains to be established.
The overall sequence conservation between catalytic domains of
phosphodiesterases that belong to the same family is >50%, while
between families, the extent of identity is less than 40% (6). When
the sequence of the catalytic domain of TbPDE2A was compared with those
of representatives of the 11 currently known mammalian PDE families,
TbPDE2A exhibited no sequence identity of more than 40% with any of
them. A comparison between TbPDE2A and class I PDEs from lower
eukaryotes, such as PDE2 of S. cerevisiae, dunce of D. melanogaster, and regA of D. discoideum
showed similarly low extents of sequence conservation.
TbPDE2A Complements PDE-deficient S. cerevisiae--
TbPDE2A was
expressed, either as the full-size enzyme or as the truncated form
without the N-terminal cGMP binding domain (amino acids 124-485), in
an S. cerevisiae strain from which both endogenous
phosphodiesterase genes had been deleted (PP5; Ref. 35). PP5 is very
sensitive to heat shock due to the absence of phosphodiesterase
activity. Transformants were tested for heat shock resistance (Fig.
4). Both the full-size enzyme and the
truncated form fully restored heat shock resistance of the indicator
strain, indicating that TbPDE2A is active in S. cerevisiae
and that the N-terminal domain is not required for the activity of the
catalytic domain. Two promotors of different strengths were used for
these expression experiments (an attenuated form of CYC1 as a weak
promotor and TEF2 as a strong promotor), but essentially identical
results were obtained. Thus, minimal amounts of TbPDE2A are apparently sufficient to rescue the heat shock resistance phenotype of the PP5
strain.
Characterization of TbPDE2A Activity--
For the characterization
of the catalytic activity of TbPDE2A, the enzyme was expressed in the
PDE-deficient yeast strain PP5, using plasmid pLT1 with the strong TEF2
promotor. TbPDE2A was expressed either as the full-length enzyme or in
its N-terminally truncated form (amino acids 124-485), which lacks the
GAF domain. To be able to monitor protein expression and stability,
both constructs contained a hemagglutinin tag at their C termini.
In vivo activity of all constructs was first assessed by
analysis of the heat shock phenotype conferred to the host strain, and
stability under assay conditions was monitored by immunoblotting with
an anti-hemagglutinin antibody.
Both constructs exhibited very similar activities with cAMP as the
substrate, with a Km in the range of 2 µM and a Vmax of 1 µmol × min Inhibitor Profile of TbPDE2A--
Inhibitor studies were performed
on lysates from PP5 expressing the full-size TbPDE2A. For the initial
screening, all inhibitors were used at a concentration of 100 µM, with a substrate concentration of 1 µM
cAMP. Only a few of the inhibitors tested exhibited a significant
effect on enzyme activity (trequinsin, ethaverine, sildenafil, and
dipyridamole), even at the high concentration used for the screen.
Several others (etazolate, erythro-9-(2-hydroxy-3-nonyl)adenine, zardaverine, pentoxyfylline, 8-methoxy-IBMX, and papaverine) inhibited the enzyme activity by about 50%. A third group, including the broad
spectrum inhibitor IBMX (rolipram, theophylline, Ro-20-1724, IBMX,
zaprinast, cilostamide, and vinpocetine), showed no significant effect
on enzyme activity. With respect to its resistance to IBMX, TbPDE2A is
similar to the mammalian PDE9 family (6). The observation that
ethaverine was significantly more effective as an inhibitor of TbPDE2A
than its parent compound papaverine was unexpected, since this
compound, the ethoxy derivative of papaverine, was so far only known as
a calcium channel blocker (53).
Subsequently, IC50 values were determined for several
inhibitors, using yeast lysates expressing the full-size construct
pTPDE2Ahm (Fig. 5). The concentration of
cAMP as substrate was set at 1 µM, i.e. the
range of its Km. Several structurally unrelated inhibitors showed similar potency against TbPDE2A, with
Ki values in the low micromolar range. The potency
of these inhibitors toward TbPDE2A is not correlated with their family
specificity for mammalian PDEs (Table
II). Trequinsin is an inhibitor of the PDE3 family; dipyridamole inhibits families 5, 6, 9, 10, and 11 (7);
and sildenafil is quite specific for family 5. Ethaverine was not known
so far as a PDE inhibitor.
The four compounds were further analyzed for their effects on cell
growth in culture. Bloodstream form trypanosomes were grown in
microtiter plates for 20 or 40 h in the presence of serial dilutions of the inhibitors (Fig. 6), and
cell proliferation was determined by an acid phosphatase-based assay
(41). All four compounds inhibited trypanosome growth with
IC50 values that were about 10-fold higher than those
determined with the soluble enzyme. The Hill slopes of the
dose-response curves were close to 1 for three of the compounds
(dipyridamole: 1.38 ± 021; sildenafil: 1.73 ± 0.69; and
trequinsin: 1.09 ± 0.63), while it was 5.19 ± 1.52 for
ethaverine. This indicates that the observed inhibition of cell
proliferation by the first three compounds is indeed due to the
inhibitory effect of the compounds on PDE activity, while the
inhibition by ethaverine may be due to the combined effects of calcium
channel blocking and inhibition of PDE activity. The results obtained
with the first three compounds indicate that the activity of TbPDE2A,
and possibly other members of this family, is essential for trypanosome
proliferation in culture.
Inhibition of TbPDE2A Raises the Intracellular cAMP
Concentration--
Inhibiting PDE activity in vivo should
raise the intracellular cAMP concentration. To verify if the inhibitors
tested above do in fact inhibit trypanosomal PDEs in vivo,
trypanosomes were incubated with sildenafil (200 µM),
trequinsin (50 µM), dipyridamole (100 µM),
or ethaverine (100 µM) for 30 and 120 min. Control cells were incubated for the same lengths of time without drug. Cell morphology and motility were normal under these conditions. Cells were
collected and processed for cAMP determination as detailed under
"Experimental Procedures." cAMP concentrations were normalized to
the values of the control cells at the respective time points. The
results given in Fig. 7 demonstrate that
trequinsin, sildenafil, and dipyridamole, but not ethaverine, lead to
an increase in the intracellular cAMP concentration, although the
extent of this increase varies between the different inhibitors. The
cAMP concentration of control cells (66 ± 14 pmol/109
trypanosomes) corresponds very well with the values obtained earlier
with another assay procedure and in different trypanosome strains
(26).
The current study reported the identification and characterization
of a member of a small family of cAMP-specific phosphodiesterases from
T. brucei. This is the first report of cloning a gene for a
phosphodiesterase from a parasitic protozoon. TbPDE2A is coded for by a
gene that represents a small family of related but different genes. DNA
sequence analysis of the locus revealed the presence of genes unrelated
to phosphodiesterases upstream and downstream of the open reading frame
for TbPDE2A, demonstrating that the genes of this PDE family are not
clustered. The open reading frame predicts a protein consisting of 485 amino acids, with a molecular mass of 55,313. The predicted start codon
is functional, as demonstrated by expression of the recombinant protein
in S. cerevisiae, and no potential extension of the open
reading frame upstream of this start codon is predicted from the DNA
sequence. The open reading frame codes for a protein with a C-terminal
catalytic domain with strong homology to class I PDEs. The extent of
sequence conservation and the inhibitor profile unambiguously classify
TbPDE2 as a new family of the class I PDEs. The N-terminal moiety
contains a single, well conserved GAF domain (32), which is separated
from the downstream catalytic domain by a linker region of about 80 amino acids. The GAF domain is very similar to those of the mammalian PDE families that contain such domains (families 2, 5, 6, 10, and 11).
TbPDE2A only contains a single GAF domain, while the mammalian PDE2,
-5, -6, and -10 all contain two such closely spaced domains. In
this respect, it most closely resembles the mammalian PDE11A (7). The
functional significance of this unusual architecture of TbPDE2A remains
to be explored. The fact that GAF domains can potentially bind cGMP may
indicate that cGMP signaling is also present in T. brucei,
lending support to an earlier claim that cGMP signaling might exist in
T. cruzi (54). The domain may serve as an integrator for
cAMP- and a cGMP-mediated signaling cascades. On the other hand, GAF
domains are representatives of a large family of domains that bind
assorted small molecules other than cGMP (32). Thus, not every domain
predicted from its amino acid sequence to be a cGMP binding domain may
actually function by binding cGMP. For instance, several
Escherichia coli proteins contain predicted cGMP-binding
domains, although E. coli does not contain a guanylyl
cyclase, and cGMP is unlikely to play a role in this organism.
Analysis of recombinant TbPDE2A demonstrated that it is a cAMP-specific
phosphodiesterase with a Km for cAMP in the 2 µM range. This Km is typical for many
of the class I PDEs. It is also in agreement with the available
estimates of the intracellular concentration of cAMP in T. brucei (1-10 µM; Ref. 26). Recombinant proteins
with or without the GAF domain exhibited similar activities with cAMP
as a substrate, and the activity of both constructs was not affected by
the presence of excess cGMP. These observations confirm that TbPDE2A is
a cAMP-specific phosphodiesterase and that cGMP either does not bind to
the GAF domain or that such a binding does not directly affect its
catalytic activity. Thus, the GAF domain may be involved in the
interactions with other components of the cell.
TbPDE2A displays a unique pharmacology that sets it apart from all
previously characterized PDE families. IBMX and theophylline, two
nonselective inhibitors of most PDEs, are not effective on TbPDE2A.
Three compounds that were found to inhibit TbPDE2A at the low
micromolar level are specific inhibitors of different mammalian PDE
families. Trequinsin (IC50 for TbPDE2A = 5.4 µM) is an inhibitor of family 3, and dipyridamole
(IC50 = 5.8 µM) is an inhibitor of the
mammalian families 5 and 6, as is sildenafil (IC50 = 9.4 µM). However, the IC50 values of trequinsin
and sildenafil for TbPDE2A are much higher than those observed with
mammalian PDEs. Furthermore, at a concentration of 5 µM,
trequinsin is likely to also inhibit PDE3 and PDE4. In conjunction, the
current data indicate a pharmacological profile of TbPDE2A that is
unlike that of any of the mammalian PDE classes. TbPDE2A is completely
insensitive to the methylxanthine inhibitors; it is inhibited by the
class 3 inhibitor trequinsin, although at rather high concentrations, while it is not affected by other class 3 inhibitors such as
cilostamide. In addition, TbPDE2A is sensitive to ethaverine
(IC50 = 14 µM), a derivative of the
nonspecific inhibitor papaverine, which exerts only marginal activity
against TbPDE2A. This finding was unexpected, since ethaverine is
pharmacologically known only as a calcium channel blocker (53).
A similar pattern of inhibition was observed when cytotoxicity was
determined with cultured bloodstream forms. Interestingly, the
dose-response curve for ethaverine showed a very steep Hill slope
(5.19 ± 1.52), indicating that the effect of this compound on
cell proliferation might be due to a combined effect of calcium channel
blockage and PDE inhibition. In contrast to ethaverine, dipyridamole
(which is not only a PDE inhibitor but also a potent inhibitor of
adenosine transporters) showed a Hill slope of around 1 (1.37 ± 0.21), with no sign of cooperative inhibition of cell proliferation. This suggests that even in the presence of dipyridamole, sufficient amounts of purines can be taken up by the trypanosomes to
allow unconstrained proliferation in culture. The presence of the
TbPDE2A inhibitors leads to a continuous increase in intracellular cAMP concentration.
The biological role of TbPDE2A is currently not known. The
identification of inhibitors of this enzyme has now provided the necessary tools for its investigation and for the experimental dissection of cAMP signaling in trypanosomes. The observation that
inhibitors of TbPDE2A prevent cell proliferation in culture demonstrates that TbPDE2A, or the TbPDE2 family as a whole, may be
essential for cell proliferation. This is also supported by the
observation that TbPDE2 mRNA is constitutively expressed. In
conjunction, these data indicate that TbPDE2A and its isoenzymes may
represent interesting targets for the development of a new generation
of trypanocidal drugs, based on phosphodiesterase inhibitors. TbPDE2A
and its relatives in T. brucei as well as in other protozoa may offer a new class of targets for the development of urgently required, novel and effective anti-protozoal drugs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EMBL4 (37),
and a 6.3-kilobase pair genomic DNA fragment was isolated and
sequenced. The fragment contained the TbPDE2 gene, flanked by an unrelated gene on either side (see "Results"). Full-length TbPDE2A for recombinant expression was constructed as
follows: The 3'-end of the open reading frame of TbPDE2A was
amplified from the cDNA plasmid pT2928 using the forward primer
pde2tyfor (5'- ATGACAATGGATGGATGTGCTTAT-3') and the reverse primer
pde2tirev (5'-CTTCTCGAGGGATCCCTATCCATGGGCAGACGAAGCCCCTGTACTC-3'), containing XhoI, BamHI, and
NcoI sites (underlined) and a stop codon
(boldface italic type). The resulting PCR
fragment (366 bp) was cloned into pGEM-T-Easy (Promega) and verified by
sequencing. The fragment was then excised by digestion with
EcoRV and XhoI and was inserted into pT2928
digested with the same enzymes. This step removed the 3'-untranslated
region and introduced an NcoI site immediately before the
stop codon and resulted in plasmid pTPDE23U.
View larger version (36K):
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Fig. 1.
Recombinant TbPDEA constructs. For
details of the constructions, see "Experimental Procedures."
Gray, N-terminal domain; dark gray,
catalytic domain; black, reconstructed C terminus of TbPDE2A
containing an NcoI site (N) for the later
insertion of immunological tags. Restriction sites are as follows:
BamHI (B); EcoRI (EI);
EcoRV (EV); NcoI (N);
NdeI (Nd); XhoI (X).
Numbering corresponds to the DNA sequence deposited in
GenBankTM (see Fig. 2), with 1770 being the first and 3224 the last nucleotide of the TbPDE2A coding region. K, Kozak
sequence and start codon. Polymerase chain reaction primers are
indicated by the horizontal arrows.
412 through
7), followed by
5'-CTAAACATGAGTCGACCTCGAGT-3' (Kozak sequence in boldface type, start-codon in boldface type and
underlined, SalI site underlined, XhoI site in
italic type). Protein expression and stability of the enzyme under
assay conditions were monitored by immunoblotting, using a monoclonal
antibody against the hemagglutinin tag (Roche Molecular Biochemicals).
70 °C. Under these conditions, TbPDE2A activity is
stable for at least several months.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 2.
Genomic organization of the TbPDE2
family. A, restriction digests of genomic DNA
hybridized with the cDNA insert of pT2928, representing the
catalytic domain and 3'-untranslated region of TbPDE2A. Restriction
enzymes used are as follows: BamHI (lane 1);
BclI (lane 2); HindIII
(lane 3); EcoRI (lane
4); EcoRV (lane 5);
PstI (lane 6); SalI
(lane 7); XhoI (lane
8). The enzymes designated by asterisks
(BamHI, HindIII, EcoRI,
PstI, and XhoI) do not cut within the fragment
used for hybridization. B, organization of the 6317-bp
genomic EcoRI fragment that contains the TbPDE2A locus.
Nucleotides 376-876 represent the RIME element. The arrows
above show the 12-bp direct repeats. Nucleotides 1770-3224
represent the open reading frame of TbPDE2A. Nucleotide 4428 is the
poly(A) addition site of TbPDE2A mRNA. Nucleotides 4693-5070
represent the open reading frame of an NHP2/RS6 homologue. The
arrows below indicate the direction of
transcription. The sequence was deposited in GenBankTM
under the accession number AF263280.
View larger version (53K):
[in a new window]
Fig. 3.
DNA sequence and predicted amino acid
sequence of TbPDE2A. GAF domain and catalytic domain are
boxed. Filled circles denote amino
acids of the catalytic domain that are conserved in at least 12 out of
14 class I PDEs (TbPDE2A, mammalian PDE1 (accession number U40372),
PDE2 (U21101), PDE3 (M91667), PDE4 (S75213), PDE5 (NM_001083), PDE6
(NM_000283), PDE7 (U68171), PDE8 (AF068247), PDE9 (AF031147), PDE10
(AF127479), D. melanogaster dunce (P12252), S. cerevisiae PDE2 (M14563), and D. discoideum RegA
(U60170). Amino acids in the gray box
(His269-Tyr281) represent the
phosphodiesterase signature motif (47).
View larger version (62K):
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Fig. 4.
Heat shock resistance. The heat
shock-sensitive PDE deletion strain of S. cerevisiae, PP5,
was transformed with plasmids containing a weak promotor (attenuated
CYC1; series 1) or a strong promotor (TEF2; series 2) and expressing
the following constructs: N-terminally truncated TbPDE2A containing a
C-terminal hemagglutinin tag (a); full-size TbPDE2A,
containing a C-terminal hemagglutinin tag (b); empty vector
(c); full-size TbPDE2A containing a C-terminal TY-1 tag
(d). A, control plate without heat shock;
B, plate with heat shock. Two or three independent clones
were tested for each construct.
1/mg (Table
I). These Km values
are well within the range of other class I PDEs. With both constructs,
cAMP hydrolysis was unaffected by the presence of a 100-fold excess of
cGMP in the reaction (data not shown). This observation defines the
catalytic activity of TbPDE2A as that of a cAMP-specific
phosphodiesterase. In addition, it indicates that cGMP either does not
bind to the GAF domain or that such a binding does not directly
influence the catalytic activity of the enzyme under the conditions of
our assay.
Comparison of Mr and enzyme parameters of full-size (TbPDE2A)
and N-terminally truncated (TbPDE2AT) phosphodiesterase
View larger version (24K):
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Fig. 5.
TbPDE2A is inhibited by inhibitors of
different structures and with specificities for different mammalian PDE
families. A, dipyridamole; B, trequinsin;
C, sildenafil; D, ethaverine; E,
example of a dose-response curve (dipyridamole).
Potency of selected PDE inhibitors
View larger version (27K):
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Fig. 6.
Cytotoxicity of selected PDE inhibitors for
bloodstream form trypanosomes. Representative examples of
IC50 determinations of PDE inhibitors against 427 bloodstream cultures. Cytotoxicity was determined after 40 h of
cell growth. A, dipyridamole; B, trequinsin;
C, sildenafil; D, ethaverine.
View larger version (17K):
[in a new window]
Fig. 7.
cAMP content of trypanosomes in the presence
of TbPDE2A inhibitors. Intracellular cAMP was determined in HCl
extracts of bloodstream trypanosomes incubated in growth medium in the
presence of inhibitors for 30 or 120 min. Control cells were incubated
without inhibitors for the same lengths of time. The figure
presents one of two independently determined, very similar data sets.
C, controls; T, trequinsin; E,
ethaverine; S, sildenafil; D, dipyridamole.
Gray boxes, 30-min incubation; black
boxes, 120 min incubation; asterisks, cAMP
concentration significantly different from control values
(p < 0.02).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Pfizer Pharmaceuticals for a generous gift of sildenafil citrate and to Phelix Majiwa (International Livestock Research Institute, Nairobi) for providing the clone pT2928.
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FOOTNOTES |
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* This work was supported by Swiss National Science Foundation Grants 31-046760.96 and 31-058927.99, by COST program B9 of the European Union Grant C98.0060, and by the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases.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/EMBL Data Bank with accession number(s) AF263280.
Recipient of a fellowship of the Ministry of Culture and Higher
Education of the Islamic Republic of Iran.
§ Present address: Microbiology and Immunology, UCLA, 405 Hildegard Ave., Los Angeles, CA 90095-1489.
¶ To whom correspondence should be addressed. Tel.: 41 31 631 46 49; Fax: 41 31 631 46 84; E-mail: thomas.seebeck@izb.unibe.ch.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M005419200
2 Naula, C., Schaub, R., Leech, V., Melville, S., and Seebeck, T. (2001) Mol. Biochem. Parasitol. 112, 19-28.
3 U. Bauer, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; bp, base pair(s).
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REFERENCES |
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