Tài liệu Báo cáo khoa học: A FYVE-containing unusual cyclic nucleotide phosphodiesterase from Trypanosoma cruzi docx

A FYVE-containing unusual cyclic nucleotide
phosphodiesterase from Trypanosoma cruzi
Stefan Kunz, Michael Oberholzer and Thomas Seebeck
Institute of Cell Biology, University of Bern, Bern, Switzerland
The cell biology of Trypanosoma cruzi, the causative
agent of South American Chagas’ disease, has been
extensively studied. Surprisingly, still very little is
known about the role of cyclic nucleotide signaling in
this organism [1]. A number of earlier studies have
indicated a role of cAMP in differentiation [2,3], and
the existence of a nitric oxide regulated guanylyl
cyclase has been suggested [4]. In T. cruzi epimasti-
gotes, a cAMP-regulated transcript has been identified
that can be induced by elevated cAMP levels [5]. The
corresponding gene, TC26, was later found to code for
an RNaseH and to be localized on a large family of
repetitive genetic elements. More recently, the T. cruzi
genome was shown to code for several adenylyl cyclas-
es, all predicted to be similarly organized, consisting of
a large N-terminal, presumably extracellular region,
which is followed by a single transmembrane helix and
a C-terminal catalytic domain [6]. The structure of
these cyclases is entirely different from that of their
mammalian counterparts, but closely similar to that of
the cyclases characterized in Leishmania donovani [7]
and in African trypanosomes [8–10]. One of these
adenylyl cyclases, TczAC, was found to interact with a
paraflagellar rod protein, and is most likely located in
the flagellum [11].
A cAMP-specific phosphodiesterase (PDE) activity
has been demonstrated in T. cruzi by various laborat-

ories [12,13]. Recently, the first cyclic-nucleotide-
specific PDE from T. cruzi has been identified and
characterized at the molecular level [14]. This enzyme,
Keywords:
Chagas’ disease; cyclic nucleotides; FYVE
domain; kinetoplastids; phosphodiesterase
Correspondence
T. Seebeck, Institute of Cell Biology,
Baltzerstrasse 4, CH-3012 BERN,
Switzerland
Fax: +41 31 6314684
Tel: +41 31 6314649
E-mail:
Website:
Nucleotide sequence data have been sub-
mitted to the DDBJ ⁄ EMBL ⁄ GenBank data-
bases under the accession numbers
AJ889575 and AJ889576 for TcrPDEC alle-
les 1 and 2, respectively.
(Received 26 August 2005, revised 20 Octo-
ber 2005, accepted 27 October 2005)
doi:10.1111/j.1742-4658.2005.05039.x
Cyclic-nucleotide-specific phosphodiesterases (PDEs) are key players in the
intracellular signaling pathways of the important human pathogen Trypano-
soma cruzi. We report herein the identification of an unusual PDE from
this protozoal organism. This enzyme, TcrPDEC, is a member of the class
I PDEs, as determined from the presence of a characteristic signature
sequence and from the conservation of a number of functionally important
amino acid residues within its catalytic domain. Class I PDEs include a
large number of PDEs from eukaryotes, among them all 11 human PDE

families. Unusually for an enzyme of this class, TcrPDEC contains a
FYVE-type domain in its N-terminal region, followed by two closely
spaced coiled-coil domains. Its catalytic domain is located in the middle of
the polypeptide chain, whereas all other class I enzymes contain their cata-
lytic domains in their C-terminal parts. TcrPDEC can complement a PDE-
deficient yeast strain. Unexpectedly for a kinetoplastid PDE, TcrPDEC is a
dual-specificity PDE that accepts both cAMP and cGMP as its substrates.
Abbreviations
DMSO, dimethyl sulfoxide; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenosin; FYVE, domain containing Fab1p, YOTB, Vac1p and EEA1PDE; GST,
glutathione-S-transferase; IBMX, isobutyl methyl xanthine; LmPDEC, phosphodiesterase from Leishmania major; PtdIns(3)P, phosphatidyl
inositol-3-phosphate; TbPDEC, phosphodiesterase from Trypanosoma brucei; TcrPDEC, phosphodiesterase from Trypanosoma cruzi.
6412 FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS
TcPDE1 is entirely cAMP-specific, and it is located
along the flagellum. In terms of its amino acid
sequence, TcPDE1 is a close homologue of the Trypano-
soma brucei PDEs TbPDE2B [15] and TbPDE2C [16],
as well as of LmPDEB1 and LmPDEB2 of Leishmania
major (Johner et al., unpublished results). All of these
enzymes belong to the class I PDEs [17]. TcPDE1 con-
tains two GAF domains [18,19] in its N-terminal moi-
ety, and a C-terminal catalytic domain. TcPDE1, as all
its other kinetoplastid homologues, is highly cAMP-
selective.
This study reports the identification and characteri-
zation of a novel and rather unusual PDE from T. cruzi.
According to the recently proposed unifying nomen-
clature for kinetoplastid PDEs [20], this enzyme was
designated as TcrPDEC. Based on the amino acid
sequence of its catalytic domain, TcrPDEC unambigu-
ously belongs to the class I PDEs. However, it is a

rather unusual PDE in several respects: (a) unlike all
other class I PDEs, its catalytic domain is localized in
the middle of the polypeptide chain, and not at its
C-terminus; (b) the N-terminal region of TcrPDEC
contains a FYVE-type domain [21,22], a functional
domain that has not been found in any PDE so far;
and (c) TcrPDEC is the first dual-substrate PDE, with
similar K
m
values for cAMP and cGMP, that has been
identified in kinetoplastids.
Results
Identification of TcrPDEC
When the T. cruzi database ( />genedb/tcruzi) was screened for putative PDEs, a gene
was identified that codes for a rather unusual PDE,
TcrPDEC (temporary gene identification number
Tc00.1047053506697.20). The open reading frame of
TcrPDEC was amplified from genomic DNA, and sev-
eral PCR products were sequenced. This analysis
revealed the presence of two distinct alleles which dif-
fer by 62 bp (out of the 2775 bp of the entire open
reading frame; 2.2% sequence divergence). These single
nucleotide polymorphisms translate into 38 amino acid
changes (4.1% amino acid substitutions; 21 conserved,
17 nonconserved). Only six of these substitutions occur
in the catalytic domain of the enzyme, and none of
them affects a residue that is crucial for function (see
below). Southern blot analysis of T. cruzi genomic
DNA by hybridization with the complete open reading
frame of TcrPDEC results in restriction enzyme pat-

terns that are compatible with the nucleotide sequence
of TcrPDEC, demonstrating that it represents a single
copy gene (Fig. 1). EcoRI, PstI and EcoRV cut once
within the open reading frame, BamHI does not cut,
and HindIII cuts three times, resulting in two frag-
ments too small to be detected by hybridization and
one fragment of two kilobases. The hybridization of
SalI-digested DNA confirmed the polymorphism of
one SalI site detected by the sequence analysis of the
two alleles. The establishment of TcrPDEC as a single-
copy gene is not entirely trivial, as large parts of the
T. cruzi genome have undergone a duplication [23].
Functional domains of TcrPDEC
The open reading frame of TcrPDEC codes for a pro-
tein of 924 amino acids (calculated relative molecular
mass of 103 169, calculated pI ¼ 5.91) with several
functional domains (Fig. 2A). The N-terminus (P
10
–
G
73
) contains a FYVE-type domain, an acronym
composed of the designations of the first four repre-
sentatives Fab1p, YOTB, Vac1p and EEA1 [21], that is
followed by two closely spaced coiled-coil regions
(D
144
–D
179
and K

207
–E
264
). FYVE-type domains are
zinc-finger-like structures, which are currently divided
10
8
6
5
4
3
2
1.5
1
0.5
EcoRI
SalI
EcoRV
BamHI PstI HindIII
kb
Fig. 1. TcrPDEC is a single copy gene. Genomic DNA of T. cruzi
hybridized with a probe representing the entire open reading frame
of TcrPDEC.
S. Kunz et al. Novel phosphodiesterase from Trypanosoma cruzi
FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS 6413
into two classes: The classical FYVE domains (exam-
ples: (HsEEA1, accession number Q15075; DmHRS,
accession number Q960 · 8; and ScVps27p, accession
number P40343) share three consensus motifs (WXXD,
R + HHCR and RVC), and they bind specifically

to membrane-embedded-phosphatidyl-inositol-3-phos-
phate [PtdIns(3)P]. FYVE-variant domains such as
HsDFCP1 and AtPRAF1 lack some of the conserved
residues (Fig. 2B), but still bind PtdIns(3)P, albeit with
lower affinity. The FYVE-related domains (e.g. rabphi-
lin 3 A (P47709) or human Rim1 (Q86UR5) exhibit
a still higher sequence divergence in the consensus
region. Their function is still undetermined. The align-
ment of the FYVE-type domain of TcrPDEC places it
close to the FYVE-variant domains. All eight cysteine
residues predicted to be involved in Zn
2+
-binding are
fully conserved (Fig. 2C), as are the two predicted heli-
cal regions. The two hydrophobic amino acids that are
inserted in the membrane upon PtdIns(3)P binding
(L
185
and L
186
in ScVps27p [22]); are represented by
L
30
and F
31
of TcrPDEC. When matched with the
WxxD R + HHCR. RVC motif of FYVE domains,
the sequence of TcrPDEC exhibits several alterations.
A glutamate residue at position four of the first block
is substituted by aspartate, arginine at the beginning of

the second block is replaced by an alanine, the two
adjacent histidine residues are replaced by serine and
glutamine, the subsequent arginine is replaced by pro-
line, and finally the arginine of the third block is
replaced by a lysine. The consequence of these replace-
ments is a decrease of the overall net charge of the
motive from +4 to +1. The effect of these changes
on a putative membrane binding of the FYVE-type
domain of TcrPDEC remains to be explored, but they
render the TcrPDEC domain unlikely to bind to
PtdIns(3)P. This prediction is confirmed by the obser-
vation that the recombinant FYVE domain of
TcrPDEC does not bind to PtdIns(3)P, nor to
PtdIns(3,4)P
2
, PtdIns(4,5)P
2
, PtdIns(3,4,5)P
3
, phos-
phatidic acid, phosphatidyl choline, phosphatidyl ser-
ine or phosphatidyl inositol in a dot-spot assay [24,25]
(data not shown).
12
3
4
AB
FYVE
DmHrs
PHD

HsKAP-1
FYVE-related
HsRIM1
FYVE-related
RnRPH3A
TcPDEC
FYVE-variant
AtPRAF1
FYVE-variant
HsDCFP1
FYVE
ScVps27p
FYVE
HsEEA1
C
Fig. 2. The FYVE-type domain of TcrPDEC. (A) Functional organization of TcrPDEC: 1, FYVE-type domain; 2 and 3, coiled-coil regions; 4, cata-
lytic domain. (B) Dendrogram of FYVE domains: TcrPDEC; HsEEA1, human early endosome antigen 1 (accession number Q15075); DmHrs,
Drosophila Hrs (Q960 · 8); ScVps27p, S. cerevisiae vacuolar sorting protein (P40343); HsDFCP1, human double FYVE-containing protein 1
(Q9HBF4); AtPRAF1, Arabidopsis PRAF1 (Q947D2); RnRPH3A, rat rabphilin-3 A (P47709); HsRIM1, human Rim1 (Q86UR5). (C) Alignment of
FYVE and FYVE-related domains. Grey boxes: the conserved Zn
2+
coordinating cysteine residues. The FYVE domain signature motifs WxxD,
R + HHCRxCG and RVC are given in bold, underlined letters. Box 1: turret loop [41]; box 2: a-helix found in the structures of HsEEA1,
DmHrs, ScVps27p and RnRabphilin-3 A. Horizontal box: putative dimer interface of EEA1 [42].
Novel phosphodiesterase from Trypanosoma cruzi S. Kunz et al.
6414 FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS
Downstream of the FYVE domain, TcrPDEC is pre-
dicted to contain two closely spaced coiled-coil regions
(S
150

–L
174
and K
207
–D
264
). These might serve to dimer-
ize the FYVE domains in a way similar to the struc-
ture that was determined for EEA1 [26].
To explore if these regions are indeed essential for
stabilizing the FYVE domain in the dimeric state, the
FYVE domain was expressed either alone (amino acids
1–74 of TcrPDEC), or in conjunction with the coiled-
coil region (amino acids 1–272). Gel filtration analysis
demonstrated that already the FYVE domain alone
migrates as a stable dimer (calculated molecular mass
8.2 kDa; apparent molecular mass upon gel filtration:
16.2 kDa) (Fig. 3A). The construct containing the two
coiled-coil regions in addition to the FYVE domain
(calculated molecular mass 30.5 kDa) eluted with an
apparent mass of 199.7 kDa, indicating the formation
of a higher order complex (Fig. 3B).
In TcrPDEC, the catalytic domain is located in the
middle of the polypeptide chain of 924 amino acids
(T
291
–S
657
; Fig. 2A). This is very unusual for a class I
PDE, as all other members of this PDE class contain

the catalytic domain in their C-terminal portions. Nev-
ertheless, the catalytic domain of TcrPDEC unambigu-
ously identifies it as a class I PDE (Fig. 4). This PDE
class includes all 11 human PDE families, and all
of its members contain the signature motif HD(LIV-
MFY)xHx(AG)xxNx(LIVMFY). Their catalytic
domains share 30–40% amino acid sequence identity
between families [17]. The overall sequence of the
TcPDE-FYVE catalytic domain conforms well with
that of other class I PDEs. It shares between 24 and
33% of amino acid sequence identity with the 11
human PDE families, and 45 and 57% sequence iden-
tity with its putative orthologs in L. major (lmjPDEC)
and T. brucei (TbrPDEC; unpublished data). All resi-
dues that have been identified as important for sub-
strate recognition, selectivity and catalysis in the
human PDEs [27,28] are conserved in TcrPDEC with
respect to HsPDE4B2 (Fig. 4). The two metal binding
sites are predicted to be formed by residues H
368
,H
372
,
H
409
,E
410
, His413, N
418
,L

438
,D
439
,E
481
,M
482
and
E
521
[28]. The hydrophobic pocket that accommodates
the purine moiety of the substrate is also conserved
and predicted to consist of Y
367
,I
522
,A
524
,S
525
,A
532
,
W
535
,L
536
,I
538
,L

539
,G
559
,S
564
,V
566
,S
569
,Q
570
, and
F
573
. Interestingly, of the two residues in this pocket
that contribute to selectivity for cAMP over cGMP
in HsPDE4, only Q
570
(corresponding to Q
443
in
HsPDE4B2) is conserved, while the residue corres-
ponding to N
395
in HsPDE4B2 is substituted by A
524
.
The side chain of this N
395
in the structure of

HsPDE4B forms two hydrogen bonds with adenine,
with the 6-NH
2
atom and with the 7-N ring atom,
while it might only form a single interaction with
either the 7-N atom or the 2-NH
2
atom of guanine.
While the presence of an N residue at this position
favors cAMP binding, it does not preclude cGMP
binding. However, in several of the human PDEs that
accept cGMP as a substrate, the position of N
395
is
substituted by an alanine (HsPDE5 and HsPDE6) or
glycine (HsPDE3 [27]). The presence of an alanine resi-
due at this position implies that TcrPDEC might be
capable of using cGMP as a substrate. This could be
experimentally confirmed (see below). Compared with
the set of 19 residues that are completely conserved
in all 11 human PDEs [27], four substitutions in
Fig. 3. Dimeric structure of the FYVE-type domain. (A) Gel filtration
(Superdex 75 PC 3.2 ⁄ 30) of an N-terminal fragment of TcrPDEC
containing the FYVE-variant domain through the coiled-coil region
(amino acids 1–272). (B) Gel filtration of FYVE-type domain alone
(amino acids 1–74 of TcrPDEC). Elution positions of the FYVE-con-
taining polypeptides are indicated by asterisks. Vertical black arrow:
elution position of TEF protease (27 kDa). Elution position of
molecular weight markers (open arrows): 1, aldolase (158 kDa); 2,
bovine serum albumin (67 kDa); 3, chymotrypsingen A (25.0 kDa);

4, aprotinin (6.5 kDa); 5, vitamin B12 (1.35 kDa).
S. Kunz et al. Novel phosphodiesterase from Trypanosoma cruzi
FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS 6415
TcrPDEC are notable. In the predicted helix 9, a con-
served alanine is substituted by S
429
in TcrPDEC. In
predicted helix 10, the first of the two vicinal histidines
is replaced by L
441
, and in predicted helix 11, a con-
served alanine is substituted by H
479
. Finally, in pre-
dicted helix 14, a conserved aspartate is replaced by
E
547
. The three substitutions represented by L
441
,H
479
and E
547
are specific for TcrPDEC. Another class1
PDE of T. cruzi, TcPDE1, that has a low K
m
and is
cAMP-selective [14], conforms to the mammalian PDE
pattern in all three positions. These three substitutions
in TcrPDEC may contribute to its dual-substrate spe-

cificity, and ⁄ or to its relatively high K
m
for both sub-
strates (see below).
Functional complementation of a PDE-deficient
S. cerevisiae strain
Deletion of the two PDE genes ScPDE1 and ScPDE2
from the S. cerevisiae genome leads to an accumula-
tion of cAMP in the cells, leading to marked heat-
shock sensitivity [29]. Heterologous complementation
of the heat-shock sensitivity phenotype of PDE-defici-
ent yeast strains has proven to be a highly sensitive
functional validation for suspected PDE genes [20–32].
The full-size open reading frame of TcrPDEC, as well
as the predicted catalytic domain (T
291
–S
657
) were
expressed in the PDE-deficient S. cerevisiae strain PP5
[31]. In addition, the same two constructs carrying
Fig. 4. Sequence alignment of catalytic domains. Grey vertical boxes indicate residues that are identical in all human PDEs as well as in
TcPDE1 and TcrPDEC; open vertical boxes indicate residues that are conserved in all 11 human PDEs and in TcPDE1, but are substituted in
TcrPDEC; black dots represent residues that are necessary for positioning the catalytically important histidine residue (arrow); boxes indicate
the consensus helices; asterisk and bold, asparagine residue which confers cAMP preference (N
395
in HcPDE4B). m, metal binding pocket;
q, Q-domain [28].
Novel phosphodiesterase from Trypanosoma cruzi S. Kunz et al.
6416 FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS

N-terminal hemagglutinin tags were also expressed.
Transformants were patched and tested for heat shock
resistance (Fig. 5). All four constructs fully restored
the heat shock resistance phenotype to the indicator
strain. The results of these complementation experi-
ments established that TcrPDEC codes for a functional
PDE, and that this enzyme can use cAMP as a sub-
strate.
Characterization of catalytic activities
Soluble cell lysates were prepared from yeast strains
expressing either full-length TcrPDEC or its catalytic
domain only. The recombinant PDE activity was
entirely dependent on the presence of divalent cations,
with Mg
2+
providing the best activity. Setting the
activity observed with 2 mm MgCl
2
as 100%, 2 mm
MnCl
2
yielded 53.5 ± 15.9% activity, 0.2 mm CaCl
2
15.3 ± 1.9%, and 30 lm ZnCl
2
19.5 ± 3.2%. The
activity obtained with 2 mm MgCl
2
was slightly, but
consistently, further stimulated by the presence of

0.2 mm CaCl
2
(116.1 ± 13.2%). In all subsequent
experiments, reactions contained 10 mm MgCl
2
.
Michaelis-Menten kinetics were determined with
cAMP or cGMP as substrates (Fig. 6A,B). TcrPDEC
exhibits a K
m
for cAMP of 31.6 ± 9 lm (N ¼ 6). This
is significantly higher than the K
m
for cAMP observed
for a previously characterized PDE from T. cruzi,
TcPDE1 [14]. Unexpectedly, TcrPDEC also hydrolyzes
cGMP with a similar V
max
, and with a K
m
of
78.2 ± 25 lm (n ¼ 3). This dual substrate specificity
might reflect the presence of the A
524
residue in the
sequence of TcrPDEC, replacing an asparagine residue
- heat shock+ heat shock
pLT-TcPDEC
pLT-TcPDEC-cat
pHA-TcPDEC

pHA-TcPDEC-cat
pLT1
pd6
Fig. 5. Reversal of the heat-shock sensitivity phenotype of S. cere-
visiae PDE deletion strain PP5. Duplicate patches of independent
transformants of S. cerevisiae PP5 incubated with (left) or without
(right) an initial heat shock at 55 °C for 15 min pLT-TcrPDEC, full-
length TcrPDEC; pHA-TcrPDEC, full-length TcrPDEC with a N-ter-
minal hemagglutinin tag; pLT-TcrPDEC-cat, catalytic domain of
TcrPDEC (W
367
-H
597
); pHA-TcrPDEC-cat, catalytic domain of TcrP-
DEC with an N-terminal hemagglutinin tag; pLT1, empty vector
(negative control); pd6, TbPDE1 (positive control).
Dipyridamole
Etazolate
%PDE civtat iy
[Inhibitor] (µM) [Inhibitor] (µM)
Trequinsin
IBMX
%P act tyDE ivi
µM cAMP
µ AMPm
o
l
µM cGMP
µm
ol GMP

D
AB
C
Fig. 6. Michaelis-Menten plots (A) with
cAMP as substrate and (B) with cGMP as
substrate. (C) and (D) show representative
plots of IC
50
determinations for various PDE
inhibitors (substrate concentration: 1 l
M
cAMP).
S. Kunz et al. Novel phosphodiesterase from Trypanosoma cruzi
FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS 6417
that appears to confer cAMP selectivity in the cAMP-
specific PDEs. This dual specificity is a novel feature
for a kinetoplastid PDE since all PDEs characterized
in these organisms so far are entirely cAMP selective
[13,15,16,30,32, Johner et al. submitted].
Inhibitor profiling
To explore the sensitivity of TcrPDEC against a spec-
trum of inhibitors, the potency of a number of com-
mercially available PDE inhibitors was determined
using 1 lm cAMP as the substrate. Most of the com-
pounds tested exhibited low potency against TcrPDEC
(Table 1), though several of them are high-potency
inhibitors of various human PDEs. Only three of the
compounds tested, trequinsin, etazolate, and dipyrida-
mole, exhibited IC
50

values below 10 lm (Figs 6C.D).
With human PDEs, these three compounds inhibit
different PDE families with high potency (trequisin:
HsPDE3, IC
50
0.0003 lm; etazolate: HsPDE4, IC
50
0.55 lm; dipyridamole: HsPDE5, IC
50
0.9 lm). All
three compounds have also proven to be effective
inhibitors of various PDE families from different kine-
toplastids (15,16,30,32, Johner et al. submitted]. The
IC
50
value of the broad-spectrum PDE inhibitor iso-
butyl methyl xanthine (IBMX) against TcrPDEC is
68 lm (Fig. 6D). This potency is similar to that found
for IBMX with many of the human PDEs. However,
it is considerably higher than what was found for other
trypanosomatid PDEs such as TcPDE1 [14], TbPDE1
[30] or TbPDE2 [16,32].
Discussion
TcrPDEC represents a novel type of class I PDE, as
defined by the sequence characteristics of its catalytic
domain [17]. All PDEs of this class exhibit a similar
overall architecture, where the N-terminal moiety con-
tains various assortments of regulatory domains and
the C-terminal part hosts the catalytic domain. Tcr-
PDEC is a notable exception to this rule, in that its

catalytic domain is localized in the middle of the poly-
peptide chain (amino acids T
291
–S
657
of a total of 924).
The C-terminal portion contains no recognizable func-
tional domains or motifs. In the amino acid sequence
of the catalytic domain, all residues that have been
identified as important for substrate recognition, selec-
tivity and catalysis [27,28] are fully conserved. In the
structure of human PDE4, two residues within the
hydrophobic purine-binding pocket were shown to be
crucial for purine binding and for adenine-specificity
(in HsPDE4B2: Q
443
and N
395
, respectively). The pres-
ence of an asparagine in this position favors the bind-
ing of cAMP, but does necessarily preclude the
binding of cGMP. However, in several of the human
PDEs that accept cGMP as their substrate, the posi-
tion of N
395
is substituted by alanine (in HsPDE5 and
HsPDE6) or by glycine (in HsPDE3) [27]. Interest-
Table 1. Potency of selected PDE inhibitors. All IC
50
values were determined at 1 lM substrate.

Inhibitor
IC
50
(lM)
Mammalian PDE selectivity (IC
50
lM)
TcrPDE TcPDE1 TbPDE2 TbPDE1
Etazolate 0.7 ± 0.04 31–127 25 PDE 4 (2)
Trequinsin 3.9 ± 6.5 13.3 2.5 PDE 3 (0.0003)
Dipyridamole 6.9 ± 4.0 17 15–27 13 PDE 5 (0.9)
PDE 6 (0.4)
PDE 8 (4.5)
PDE 10 (1.1)
PDE 11 (1–2)
Papaverine 25 111 304 30 n.s. (5–25)*
IBMX
†
68 >1000 >1000 >1000 n.s. (2–50)*
MM-IBMX
‡
>75 >100 PDE 1 (4)
EHNA >100 >100 >100 >100 PDE 2 (1)
Milrinone >100 >100 PDE 3 (0.3)
Cilostamide >100 >100 >100 PDE 3 (0.005)
Zardaverine >100 >100 PDE 3 (0.5)
PDE 4 (0.2–0.8)
Rolipram >100 > 500 >100 >200 PDE 4 (2)
Ro20-1724 >100 >100 >100 PDE 4 (2)
Pentoxifylline >100 >800 n.s.* (45-150)

*n.s., nonselective inhibitor;
†
isobutyl methyl xanthine;
‡
8-methoxymethyl-IBMX; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenosin.
Novel phosphodiesterase from Trypanosoma cruzi S. Kunz et al.
6418 FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS
ingly, the position corresponding to N
395
in
HsPDE4B2 is occupied by an alanine residue (A
524
)in
TcrPDEC. Based on the information available from
the human PDEs, this finding predicts that TcrPDEC
could accept cGMP as a substrate. This prediction
could be experimentally verified. Recombinant TcrP-
DEC proved to be a dual-specificity PDE, with K
m
values of 32 lm for cAMP and 78 lm for cGMP,
respectively. These K
m
values are relatively high when
compared with most of the human and trypanosomal
PDEs. Nevertheless, they are similar to those of the
human dual-specificity PDE HsPDE2 (30–50 lm for
cAMP and 15–30 lm for cGMP [33]).
While the observation that TcrPDEC can hydrolyze
cGMP is in perfect agreement with the prediction from
its amino acid sequence, the biological significance of

this finding is not at all clear. Paveto et al. have pre-
sented evidence that T. cruzi might contain a cGMP
signaling pathway that can be stimulated by nitric
oxide [4]. In other kinetoplastids such as L. major or
T. brucei, no cGMP PDE activity was detectable in
whole cell lysates (unpublished data). Database screen-
ing of kinetoplastid genomes has not so far identified
any genes for putative soluble guanylyl cyclases, as
would have been predicted by the work of Paveto
et al. [4]. However, one cannot exclude that one or
several of the predicted adenylyl cyclases identified in
the T. cruzi and other kinetoplastid genomes might
actually function as a guanylyl cyclase. In mammalian
adenylyl and guanylyl cyclases, a single amino acid
substitution can determine the substrate specificity [34].
A second unusual feature of TcrPDEC is the pres-
ence of a FYVE-type domain at its N-terminus. FYVE
domains have been identified in numerous proteins,
but so far never in a PDE. While some FYVE
domains of some proteins were shown to interact with
PtdIns(3)P and to locate to endosomal membranes, the
functional role of the FYVE-variant and FYVE-rela-
ted domains is less clear. The FYVE-type domain of
TcrPDEC is followed by two closely spaced coiled-coil
regions. Such coiled-coil domains were shown to serve
to dimerize the FYVE domains in EEA1 [26]. In con-
trast, gel filtration analysis of the FYVE-type domain
of TcrPDEC has now shown that it can form a stable
dimer in the absence of the coiled-coil domains. This
dimerization might enhance the interaction of the

FYVE-type domain with a target membrane. On the
other hand, the dimerization will also entail a dimeri-
zation of the downstream catalytic domains, and may
thus influence their catalytic properties. The coiled-coil
region of TcrPDEC may mediate an additional level
of organization, the function of which remains to be
explored. The overall concept of dimerization and
possibly modulation of the activity of PDE catalytic
domains via their regulatory N-terminal regions has
been established for human HsPDE2 and HsPDE5,
where dimerization takes place via GAF domains [35].
A similar GAF-mediated dimerization and modulation
also occurs in TbPDE2B of T. brucei [36] and possibly
also in TbPDE2C.
The overall sequence of the FYVE-type domain of
TcrPDEC exhibits a smaller overall electrical charge
than the canonical FYVE domains. The functional
consequences of this are not clear, as we have not been
able to demonstrate binding of a recombinant FYVE-
type domain of TcrPDEC to individual phospholipids
in a spot assay. While this naive approach works with
certain FYVE domains [24,25], it is certainly less than
definitive, since the binding requirements for individual
FYVE domains may be more complex than just indi-
vidual phospholipids spotted on a solid surface. If the
FYVE-type domain of TcrPDEC does in fact interact
with membranes, it may serve to confer a precise sub-
cellular localization to the enzyme. This is a most
interesting possibility, as the role of subcellular local-
ization assumes an ever greater significance in the

emerging concepts of intracellular signal transduction
[37]. In fact, being in the right place at the right time
may be the preeminent requirement for the proper
working of a signaling enzyme such as a PDE.
Experimental procedures
Materials
Radiochemicals were purchased from Moravek Biochemi-
cals Inc (Hartmann Analytik, Zurich, Switzerland). cAMP
and cGMP (sodium salts) were obtained from Sigma
(Buchs, Switzerland). PDE inhibitors were from the follow-
ing sources: I IIBMX, papaverine, milrinone, trequin-
sin, 8-methoxymethyl-IBMX, dipyridamole and rolipram
were from Sigma; erythro-9-(2-hydroxy-3-nonyl)adenosin
(EHNA), zardaverine, cilostamide and Ro-20–1724 were
from BioMol (Anawa Trading, Wangen, Switzerland); etaz-
olate and pentoxifylline were from Calbiochem (Juro Sup-
plies, Lucerne, Switzerland). DNA sequencing reactions
were run with BigDye terminators (PE-Biosystems) and
were analyzed on an ABI Prism 377 instrument. Genomic
DNA was extracted from the T. cruzi promastigotes sup-
plied by R. Brun, Swiss Tropical Institute, University of
Basel.
Identification and cloning of TcPDE-C
A search of the T. cruzi database ( />genedb/tcruzi) for PDE-coding sequences identified an open
S. Kunz et al. Novel phosphodiesterase from Trypanosoma cruzi
FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS 6419
reading frame (Tc00.1047053506697.20) that appeared to
code for a highly unusual PDE, termed TcrPDEC. Its
entire coding region was amplified by high-fidelity PCR
(Expand High FidelityÒ PCR System, Roche Diagnostics,

Rotkreuz, Switzerland) using T. cruzi genomic DNA as the
template. The primers used were TcPDE4 for (5¢-CAG
TCGACATATGTCGGAGGACGCTGGGCTTC-3¢) and
TcPDE4-rev (5¢-CGTGGATCCTCAGCACTGCGTCAA
CAGAGTG-3¢). The PCR product was cloned into the
pCR2.1-TOPO vector. The predicted catalytic region of
TcrPDEC (T
291
–S
657
) was amplified separately, using prim-
ers TcPDE4-catf (5¢-CAGTCGACATATGACAATACT
CGCAGTTGTTCC-3¢) and TcPDE4-catr (5¢-CTGGAT
CCTCAACTGGCTGTTCTCAGCTCCTG-3¢) and cloned
into pGEM-T Easy plasmid vector (Promega, Catalys,
Wallisellen, Switzerland). All cloned PCR products were
verified by DNA sequencing.
Expression in S. cerevisiae
For expression in S. cerevisiae, the entire open reading
frame and the catalytic region (T
291
–S
657
) of TcrPDEC were
cloned via SalI and BamHI restriction sites into two variants
of the yeast expression vector pLT1 [30]. One variant directs
the expression of the genuine protein, while the other adds
an N-terminal hemagglutinin-tag to facilitate detection of
the recombinant proteins. Transformation of the constructs
into the PDE-deficient S. cerevisiae strain PP5 (MATa leu2–

3 leu2–112 ura3–52 his3–532 his4 cam pde1::ura3 pde2::
HIS3) was carried out as described earlier [30,31].
Complementation assay
The heat-shock assay to detect complementation of the
PDE-deficient phenotype of the S. cerevisiae strain PP5 was
carried out exactly as described [30]. Single yeast colonies
were replica patched onto YPD plates (10 gÆL Bacto-Yeast
extract, 20 gÆL Bacto-Peptone, 20 gÆL glucose, 20 gÆL bacto-
agar) prewarmed to 55 °C, and were incubated for another
15 min at 55 °C. Plates were then cooled to room tempera-
ture and incubation was continued at 30 °C for 18–36 h.
Yeast cell lysis
Yeast cell lysis was performed as described [32] with minor
modifications. Briefly, yeast cells grown to mid-log to end-
log phase in SC-leu medium were collected, resuspended in
the original volume of prewarmed YPD medium, and incu-
bated for an additional 3.5 h at 30 °C to maximize protein
expression. Cells were washed twice in H
2
O, and the washed
cell pellet was stored at )70 °C. The pellet was thawed on
ice and suspended in an equal volume of ice-cold extraction
buffer [50 mm Hepes pH 7.5, 100 mm NaCl, 1 · Complete
Ò
protease inhibitor cocktail with EDTA (Roche Diagnos-
tics)]. Cells were lysed by grinding with glass beads (0.45–
0.50 mm) in 2 mL Sarstedt tubes, using a FastPrep FP120
cell disruptor (3 · 45 s at setting 4). The subsequent centrif-
ugation steps were carried out exactly as described. Glycerol
was added to the resulting supernatant to a final concentra-

tion of 15% (v ⁄ v), aliquots were snap-frozen in liquid nitro-
gen and were stored at )70 °C. Protein expression and
stability of the enzyme under assay conditions were monit-
ored by immunoblotting, using a monoclonal antibody
against the hemagglutinin tag (Roche Diagnostics).
Protein–lipid overlay assay
Protein–lipid binding studies were performed as described
[38], with the following modifications: Lipids were dissolved
in chloroform ⁄ methanol ⁄ water (1 : 2 : 0.8 by volume). A
20–2500-pmol amount of each lipid were spotted onto
nitrocellulose filters and were air-dried for 1 h. Membranes
were blocked with 3% (w ⁄ v) fat-free bovine serum albumin
(Sigma) in 10 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 0.1%
(v ⁄ v) Tween-20) for 1.5 h at room temperature. Filters were
incubated with 2 lgÆmL
)1
of recombinant GST-fusion pro-
teins overnight at 4 °C. After washing the filters five times
with incubation buffer, the bound GST fusion protein was
quantitated by developing the filters with anti-GST anti-
body, followed by horse radish conjugated anti-IgG anti-
body.
Phosphodiesterase assay
PDE activity was determined by a modification of the two-
step procedure of Thompson and Appleman [39] as des-
cribed previously (Johner et al. submitted). PDE activity
was determined in 50 mm Hepes pH 7.5, 0.5 mm EDTA,
10 mm MgCl
2
,50lgÆmL

)1
bovine serum albumin in a final
volume of 100 lL. Each assay contained 50 000 cpm
3
H-labelled cAMP or cGMP, with unlabeled cAMP or
cGMP added to the desired total substrate concentration.
Reactions were run at 30 °C for 15 min. They were then
stopped by the addition of 25 lL of 0.5 m HCl, neutralized
with 20 lL1m Tris base, and digested with 10 lL of calf
intestinal alkaline phosphatase (Roche Diagnostics; 1 unit
per 10 lL) for 15 min at 37 °C. After dephosphorylation,
the reactions were applied to 1 mL columns of QAE-Sepha-
dex A25 in 30 mm ammonium formate, pH 6.0. The
3
H-adenosine or
3
H-guanosine formed during the reaction
was eluted with 1.6 mL of 30 mm ammonium formate,
pH 6.0 into 3.5 mL water-miscible scintillation fluid (Pack-
ard Ultima Flo; Packard Bioscience, Zurich, Switzerland).
In all reactions no more than 20% of the substrate was
hydrolyzed. Assays were always carried out in triplicates,
and at least three independent experiments were performed.
IC
50
studies were all carried out at 1 lm substrate concen-
tration. Inhibitors were dissolved in dimethyl sulfoxide.
Novel phosphodiesterase from Trypanosoma cruzi S. Kunz et al.
6420 FEBS Journal 272 (2005) 6412–6422 ª 2005 The Authors Journal compilation ª 2005 FEBS
The dimethyl sulfoxide (DMSO) concentration in the

final assay solutions never exceeded 1%, and appropriate
control reactions with DMSO alone were always included.
Data were analyzed using the GraphPad Prism software
package.
Gel filtration analysis of the FYVE-variant domain
PCR-amplified DNA fragments corresponding to amino
acids 1–74 of TcrPDEC (FYVE-variant domain alone) and
1–272 (FYVE-variant domain plus coiled-coil region) were
cloned into the expression vector pGST-parallel2 [40].
The recombinant proteins are N-terminally fused to
glutathione-S-transferase, from which they are separated by
a TEV protease cleavage site. Recombinant protein from
125 mL bacterial culture were purified by batch-adsorption
to 125 lL Glutathione-Uniflow resin (BD Biosciences,
Basel, Switzerland) for 2 h at 4 °C. The resin was washed
four times with ice-cold 50 mm Tris ⁄ HCl, pH 7.5, 150 mm
NaCl, 10 mm 2-mercaptoethanol, 5 mm sodium citrate,
10 lm ZnCl
2
and 5% glycerol. After washing, the beads
were suspended in an equal volume of washing buffer,
30–50 units of AcTEV protease (Invitrogen; Juro Supplies,
Lucerne, Switzerland) were added, and the slurry was incu-
bated for 36 h at 6 °C on a rotating wheel. The beads were
then pelleted by centrifugation, and the supernatant con-
taining the released FYVE-variant domain was purified by
centrifugation through a 0.2 lm filter.
Proteins were analyzed on a Superdex 75 PC 3.2 ⁄ 30 gel
filtration column, using the Pharmacia Smart system. The
column was precalibrated with the following markers: aldo-

lase (158 kDa), bovine serum albumin (67 kDa), ovalbumin
(44 kDa), chymotrypsinogen A (25 kDa), RNase A
(13.7 kDa), aprotinin (6.5 kDa) and vitamin B12 (1.35 kDa).
All fractions were subsequently analyzed by gel electrophor-
esis. The larger FYVE-variant ⁄ coiled-coil polypeptide was
further analyzed on a Superdex 200 PC 3.2 ⁄ 30 column calib-
rated with the same markers, plus the following additional
ones: thyroglobulin (670 kDa), ferritin (450 kDa) and cata-
lyze (232 kDa).
Acknowledgements
We would like to thank M. Linder and X. Lan Vu for
their technical assistance, R. Brun of the Swiss Trop-
ical Institute, University of Basel, for T. cruzi cultures,
the laboratory of J. H. Hurley (NIH, Bethesda, MD,
USA) for providing expression plasmids for Vps27p-
FYVE-GST fusion proteins, Peter Buetikofer (Institute
of Molecular Biology, University of Bern) for provi-
ding phospholipids and valuable advice. This work
was supported by grant Nr 3100–067225 of the Swiss
National Science Foundation, and by the COST B22
programme of the European Union.
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