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: thomas.seebeck@izb.unibe.ch Website: http://www.izb.unibe.ch 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 (http://www.genedb.org/ 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 (http://www.genedb.org/ 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. 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