molecular cloning and expression of human cgmp-binding cgmp-specific phosphodiesterase (pde5)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247, 249–254 (1998)ARTICLE NO. RC988769

Molecular Cloning and Expression of HumancGMP-Binding cGMP-SpecificPhosphodiesterase (PDE5)1

Peter Stacey, Stuart Rulten, Alison Dapling, and Stephen C. Phillips2

Department of Molecular Pharmacology, Pfizer Central Research, Sandwich, Kent CT13 9NJ, UK

Received May 4, 1998

are currently subdivided into 7 major families [1,2].A human PDE5 cDNA has been isolated which con- Each family, and even members within a family, ex-

tains an open reading frame encoding an 875 amino hibit distinct tissue, cell and subcellular expressionacid, 100,012 Da polypeptide, the expression of which patterns and hence participate in discrete signal trans-yields a protein of the predicted size and is capable of duction pathways [1]. The PDE5 (cGMP-bindinghydrolyzing cGMP. The deduced amino acid sequence cGMP-specific PDE) family is of particular interest dueis very similar (95%) to that of bovine PDE5, and com- to its involvement in the NO/cGMP signaling pathway,prises a conserved cGMP-binding domain and cata-

which modulates smooth muscle tone [3], and the de-lytic domain. Northern analysis reveals a major andvelopment of sildenafil (VIAGRAy, UK-92,480), anminor transcript ofÇ9 kb andÇ8 kb respectively, thusorally active PDE5 inhibitor. Sildenafil is efficaciousindicating the existence of at least two splice variants,in the treatment of male erectile dysfunction [4] bythe major form being readily detected in bladder, co-potentiating NO mediated increases in cGMP in corpuslon, lung, pancreas, placenta, prostate, small intestine,cavernosal smooth muscle [5].and stomach. q 1998 Academic Press

PDE5 was first purified and characterized from rat [6]and bovine [7] lung, with enzyme activity also beingshown to be present in a variety of other tissues includingplatelets, spleen, and vascular smooth muscle [8,9,10].The cyclic nucleotides cAMP and cGMP function as Bovine PDE5 is specific for cGMP hydrolysis with thesecond messengers for a plethora of extracellular sig- native protein having a moderate affinity for cGMP (Km

nalling molecules, such as neurotransmitters and hor- Å 5.6 mM) and no significant hyhrolytic activity againstmones, and mediate their effects by interaction with a cAMP [7]. Protein sequencing and molecular cloning sub-variety of intracellular targets, e.g., kinases, ion chan- sequently lead to the isolation of a PDE5 cDNA fromnels and transcription factors. Their intracellular lev- bovine lung which encoded a protein of 865 AA residuesels are regulated through a dynamic balance of the [11] - denoted as BTPDE5A1 in accordance with stan-rates of synthesis by cyclases and degradation by cyclic dardized nomenclature [2]. More recently, a rat PDE5nucleotide phosphodiesterases (PDEs). cDNA has been described which appears to be a distinct

PDEs form a superfamily of enzymes which catalyse splice variant, i.e., PDE5A2 [12].the hydrolysis of 3 *:5*-cyclic nucleotides to the corre- PDE5 comprises a C-terminal catalytic domain ofsponding nucleoside 5*-monophosphates. On the basis Ç250 AA residues, the sequence of which is well con-of their substrate specificities, kinetic properties, regu- served between all mammalian PDEs [13], and an N-latory features and AA sequences, the various PDEs terminal cGMP-binding domain; this is supported by

the finding that proteolytic digestion of bovine PDE5liberates two distinct domains [7]. The cGMP-binding1 The nucleotide sequence reported in this paper has been submit-domain of PDE5 spans Ç380 AA residues and is alsoted to the GenBank/EMBL Data Bank with Accession Numbera conserved feature of the PDE6 (photoreceptor PDE)AJ004865.

2 Corresponding author. Fax: /44 (0)1304 615600. E-mail: and PDE2 (cGMP-stimulated PDE) families [14]. [email protected]. domain constitutes a distinct cGMP-binding element

Abbreviations: AA, amino acid; bp, base pair; Da, Dalton; EDTA, which bears no significant sequence homology to that ofethylene diamine tetraacetic acid; HEPES, N-(2-hydroxyethyl)piper- other cGMP-binding proteins, e.g., the catabolite geneazine-N*-(2-ethanesulfonate); PAGE, polyacrlyamide gel electropho-

activator protein family [15,16], nor the guanine nucle-resis; PCR, polymerase chain reaction; PDE, phosphodiesterase;SSC, saline sodium citrate buffer; SDS, sodium dodecyl sulfate otide binding domain of G-proteins [17].

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 247, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on both strands, the encoded AA sequence being: MDYKDDDDKGS-We report here the cDNA cloning of a functionallyRSEARGIQRRAG-human PDE5A1 (Fig. 1; FLAG tag and linker-active human PDE5, HSPDE5A1, and an analysis ofderived sequence underlined). Recombinant viral stocks were pre-its deduced AA sequence and its expression in a range pared using the Bac-toBac system (Life Technologies) according to

of tissues. the manufacturer’s protocol, and Sf9 cells were cultured in Sf 900 IIserum-free media (Life Technologies) at 277C. For expression, 1 1108 cells in 100 ml were infected at a multiplicity of infection of 1.MATERIALS AND METHODS Cells were harvested 48 hours post-infection for assay.

PCR Isolation of a Partial Human PDE5 cDNA Northern Blotting AnalysisA sense (5*-GGCATCGTGGGMCAYGTSGCM-3 *) and antisense A HindIII/PstI fragment encoding the catalytic domain of human

primer (5*-GACAGCTCAAAGTCRCTGAAGYKRAA-3 *) correspond- PDE5A was labeled with [a-32P]dCTP using a Megaprime kit (Amer-ing to the cGMP-binding and catalytic regions respectively were sham), and reaction products (probe) purified using Chromaspin-30used. PCR reactions containedÇ10 ng human lung cDNA (Clontech, columns (Clontech). Multiple Tissue Northern blots were purchasedPalo Alto, CA), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, (Clontech), prehybridized in ExpressHyb (Clontech) at 687C for 60.2 mM dNTPs, 2.5 units of Taq DNA polymerase (Perkin-Elmer, hours and hybridized (Ç1 1 106 cpm probe/ml) at 687C for 16 hours.Norwalk, CT) and each primer at 300 nM. Two rounds of amplifica- Blots were washed at 3 1 SSC, 1% (w/v) SDS at 457C followed by 1tion were carried out: 957C/1.5 min, 507C/1.5 min, 727C/3 min, 34 1 SSC, 1% (w/v) SDS at 457C and exposed to a phosphorimagercycles; 957C/1.5 min, 507C/1.5 min, 727C/7 min, 1 cycle. PCR products screen (Molecular Dynamics, Sunnyvale, CA) overnight. Blots werewere subcloned into pUC18 using the Sureclone Ligation Kit (Phar- checked for equal loading of poly(A)/ RNA in each lane using a hu-macia Biotech, Uppsala, Sweden), and plasmid DNA prepared (Qia- man b-actin cDNA probe as described above (data not shown).gen, East Sussex, UK). All DNA sequencing was performed on bothstrands by fluorescence-tagged dye terminator cycle sequencing (Per-

Western Analysiskin-Elmer) and analyzed on an ABI 373A DNA sequencer (AppliedBiosystems, Foster City, CA).

PDE5A1 infected and mock infected cell lysates (Ç1 1 104 cellequivalents) were separated by denaturing PAGE using the Nu-

cDNA Library Screening PAGE mini-gel system (Novex, San Diego, CA) and either stainedwith coomassie or transferred to a polyvinylidene difluoride mem-

Approximately 1 1 106 plaque forming units from both a human brane (Novex) for immunoblotting. Western analysis was performedprostate and human skeletal muscle lgt10 cDNA library (Clontech) by enhanced chemiluminescence (Amersham) according to the manu-were screened by hybridization using standard procedures [18]. The facturer’s protocol, using an anti-FLAG antibody (Sigma, Dorset,hybridization probe was generated by labeling the human PDE5 par- UK) and a horse radish peroxidase conjugated anti-mouse IgG (Bio-tial cDNA with [a-32P]dCTP using a Megaprime kit (Amersham, Rad, Herts, UK) as a secondary antibody at 1:500 and 1:750 dilutionsBucks, UK) and purifying the reaction products with Chromaspin- respectively.30 columns (Clontech). Purified, positive l phage were analyzed bysequencing, i.e., phage were used as a template in PCR reactions

Enzyme Preparation and Phosphodiesterase Assaycontaining a lgt10-forward primer (5*-CGAGCTGCTCTATAG-ACTGC-3 *) and a lgt10-reverse primer (5*-GGGTAAATAACAGAG-

Transfected Sf9 cells were harvested by centrifugation (1,000 1 gGTGGC -3 *) at 1 mM with 30 rounds of amplification: 947C/10 min;for 10 min), resuspended in homogenization buffer (20 mM HEPES557C/1 min, 727C/4 min, 947C/30 sec, 30 cycles; 557C/1 min, 727C/10pH 7.2, 1 mM EDTA, 20 mM sucrose, 150 mM NaCl and 1 proteasemin, 1 cycle. Each PCR product was directly sequenced from bothinhibitor tablet (Boehringer) per 50 ml) at 1 1 107 cells/ml and dis-ends using nested lgt10 primers (5*-ATGAGTATTTCTTCCAGGGT-rupted by sonication. Cellular debris was removed by centrifugation3 * and 5*-TGAGCAAGTTCAGCCTGGTT-3 *). Selected clones wereat 14,0001 g for 10 min and supernatant stored in aliquots at0707C.further characterized by preparing phage DNA [18] and subcloningPDE activity was measured using a modification of the method ofthe inserts into pBluescript KS/ (Stratagene, La Jolla, CA) for se-Hurwitz et al. [20], i.e., in assay buffer containing 40 mM Tris-HClquencing and subsequent subcloning steps. The sequence of thepH 7.4, 10 mM MgCl2 and 2 mg/ml bovine serum albumin (finalclones used to derive a consensus for PDE5A1 was determined onconcentrations) with 0.5 mM [3H]cGMP (Amersham) as substrate inboth strands.a final volume of 100 ml. All assays were performed in triplicate.

Subcloning and Expression of Human PDE5A1RESULTS AND DISCUSSION

A composite clone which encoded the full length protein with anN-terminal epitope tag was constructed from 2 clones (Fig. 1) for A CLUSTAL alignment [21] of bovine PDE5 withexpression in insect cells. The C-terminal half was isolated by PCR

members of the PDE6 family revealed 2 conserved re-using a sense primer (5*-GCGAATTCAAGCTTTTGTCATCTTTTG-gions around the cGMP-binding and catalytic domains,TGGC-3 *) covering a unique HindIII restriction site (Fig. 1) and an

antisense primer (5*-GCTCTAGATTATTAGTTCCGCTTGGCCTGG- GIVGHVAA and FSFSDFELS respectively, whichCCGCTTTCC-3 *) at the stop codon, the latter incorporated a tandem were used to design 2 partially degenerate PCR prim-stop codon and unique XbaI restriction site to facilitate subcloning. ers. These primers (Fig. 1) were used to isolate a partialPCR was performed using the Expand High Fidelity PCR system

human PDE5 cDNA from lung, the sequence of which(Boehringer Mannheim, West Sussex, UK) and the following cycleexhibited 94% identity over the entire 1,020 bp to theconditions: 947C/5 min; 507C/1 min, 727C/1min, 947C/1 min, 30 cycles;

507C/1 min, 727C/10 min, 1 cycle. The N-terminal half, minus the corresponding sequence of bovine PDE5 [11] (data notfirst two AA residues, was isolated as a BsrBI/HindIII fragment shown). The partial human PDE5 cDNA was then used(Fig. 1) and ligated with the HindIII/XbaI fragment into the StuI/ as a hybridization probe to screen human prostate andXbaI sites of the baculovirus transfer vector pFASTBAC (Life Tech-

skeletal muscle cDNA libraries. This resulted in thenologies, Gaithersburg, MD) which had been modified to include a5* FLAG epitope tag [19]. The sequence of the insert was determined isolation of several PDE5 cDNA clones, 2 of which to-

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FIG. 1. Nucleotide sequence (top) and deduced AA sequence (bottom) of human PDE5A1. The consensus sequence shown is encoded bytwo cDNA clones which span residues 1-2137 and 364-3041 (arrowed brackets), and the AA sequence of the catalytic domain is highlightedin bold text. The PCR primers used to isolate the partial human PDE5 cDNA are indicated (arrows), along with the restriction enzymesites for BsrBII, HindIII and PstI (5* to 3 *; boxed sequences) which were used for subcloning and/or generating probes.

gether covered the entire coding region (Fig. 1) and tide. Given the high degree of similarity (95%) to theAA sequence of bovine PDE5 (Fig. 2), even at the N-were used for subsequent subcloning for expression.

Sequencing of the human PDE5 cDNA clones lead to and C-termini, we propose that this cDNA encodes thehuman homologue hence is denoted as HSPDE5A1 inthe assembly of a consensus nucleotide sequence (Fig.

1) and the identification of a 906 AA long open reading accordance with standardized nomenclature [2].A BLAST2 alignment [23] of human PDE5A1 withframe which could extend further in the 5* direction.

However, an alignment of the deduced AA sequence the other known PDE5 AA sequences, i.e., bovine [11]and rat [12], reveals a high degree of conservation, 95%with that of bovine PDE5 (Fig. 2.) suggests that the

initiation codon is in fact the first methionine encoun- and 93% respectively, except at the N-terminus wherethe rat sequence differs significantly (Fig. 2). Presum-tered (nucleotide position 94; Fig. 1). This is further

supported by the occurrence of an identical match to ably, the latter represents a distinct splice variant forwhich a human equivalent may exist. The functionalthe consensus Kozak sequence at this position, i.e.,

ACCATGG [22]. In addition, the predicted coding se- significance of PDE splice variants is now beginning tobe examined with evidence suggesting potential rolesquence of 875 AA (Fig. 1) would yield a 100,012 Da

polypeptide which is in close agreement with the in regulating enzyme stability and, perhaps more cru-cially, both the activity of the enzyme and its locationknown molecular weight of native bovine PDE5 [7].

Therefore, we conclude that the human cDNA de- in the cell by conferring an ability to associate withspecific cellular membranes [24].scribed here encodes an 875 AA, 100,012 Da polypep-

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FIG. 2. Amino acid sequence alignment of human PDE5A1 with bovine PDE5A1 (accession L16545) and rat PDE5A2 (accession D89093).Identities and similarities are indicated below the sequences by asterisks (*) and dots (.) respectively. The cGMP-binding domain (upper)and catalytic domain (lower) are boxed, and the phosphorylation site (arrow), tandem cGMP-binding motifs (bold text), and catalytic domainresidues conserved between all known mammalian PDE families (bold, underlined asterisks) highlighted.

At the C-terminus of human PDE5A1 is a catalytic spans Ç380 AA residues (Fig. 2). Overall this domainexhibits 97% and 93% similarity to that of bovine anddomain of Ç250 AA residues, this sequence being well

conserved between all mammalian PDEs [13], which rat PDE5 respectively, with human PDE6 having 27%similarity and human PDE2 26%. Within the cGMP-contains the PDEase signature motif HDX2HX4N [25].

The degree of similarity across this region for bovine binding domain of human PDE5A1 are tandem repeatsof the conserved sequence motif N(K/R)XnFX3DE [11]and rat PDE5 is 97% and 95% respectively, followed

most closely by human cone PDE6 (44%), which sup- (Fig. 2), the NKXnD motif having been shown by muta-genesis to be important for cGMP binding [28].ports the assignment of this human PDE as PDE5A.

Within this domain are 21 AA residues which are abso- For bovine PDE5 it has been demonstrated that thecGMP-binding domain contains two kinetically distinctlutely conserved between all known mammalian PDE

families (Fig. 2) [26], therefore, they almost certainly sites, site a and site b, which have high and low dissoci-ation constants for cGMP respectively [29] - Scatchardplay an important role in cyclic nucleotide binding and/

or hydrolysis. Interestingly, it has been suggested that analysis suggests that this is also the case for PDE2and PDE6 [30,31]. Furthermore, it has been shown thatthe HX3HX24E motif which exists in tandem in mam-

malian PDEs, including human PDE5A1, functions to cGMP binding to PDE5 enhances phosphorylation at asingle serine residue both in vitro [7] and in intactcoordinate Zn2/ in the catalytic site similar to that of

Zn2/ hydrolases such as thermolysin [27]. This ac- cells [32], and that both cGMP-binding sites need to beoccupied for phosphorylation [33]. The phosphorylationcounts for 5 of the 21 AA residues; the glutamic acid

residue at the end of the first repeat is either substi- site in bovine PDE5, serine-92, is also conserved inhuman PDE5A1 and rat PDE5 (Fig. 2), and is knowntuted for by aspartic acid or there is a nearby glutamic

acid in PDEs where this motif is not conserved. How- to be phosphorylated by both cGMP-dependent andcAMP-dependent protein kinases [7]. However, despiteever, the precise function of this motif and that of the

remaining residues awaits further study. these studies, the functional significance of cGMP bind-ing to this allosteric site and phosphorylation have yetLike other cGMP-binding PDEs, human PDE5A1

comprises an N-terminal cGMP-binding domain which to be elucidated since neither have an effect on the

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possibly in intact cells or crude systems due to the pos-sible involvement of additional regulatory factors.Analysis of the AA sequence of human PDE5A1 usingPROSITE [36] did not reveal any additional proteinmotif that could be ascribed with confidence as beingfunctionally significant.

Northern analysis was performed on poly(A)/ RNAfrom a range of human tissues using the catalytic do-main of human PDE5A1 as a probe. The data obtainedindicate that PDE5A is expressed in the tissues exam-ined as a major transcript of Ç9 kb and a less promi-nent transcript of Ç8 kb (Fig. 3), which suggests thatat least two PDE5A splice variants exist. Given thecoding region spans 2,625 bp, the 5* and/or 3 * untrans-lated regions of the corresponding transcript are largeand are not represented in the clones we isolated whichresulted in a 3,041 bp composite cDNA (Fig. 1). Appre-ciable expression of PDE5A mRNA was observedin bladder, colon, lung, pancreas, placenta, prostate,small intestine and stomach, with low level expressionFIG. 3. Northern blotting analysis of PDE5A expression in hu-in brain, heart, kidney, skeletal muscle and uterus andman tissues. Approximately 2 mg poly(A)/ RNA per tissue (as indi-no detectable transcript in liver (Fig. 3) - low levels ofcated above lane) was probed with the catalytic domain of human

PDE5A. Size markers are indicated left of panel. PDE5 mRNA could result from blood vessels which areknown to contain PDE5 activity [10]. These data areconsistent with a role for PDE5 in smooth muscle.

Finally, the ability of the human PDE5A1 cDNA tokinetic properties of the enzyme [28,33]. It has beenyield a functionally active protein of the predictedsuggested that the cGMP-binding sites of PDE5 servesize was examined by expressing an epitope taggedas a buffer to protect submicromolar levels of cGMPconstruct in insect cells. The PDE5A1 expression con-from hydrolysis [34] and that cGMP binding might in-struct resulted in the production of an Ç100 kDaduce a subtle conformational change, possibly via phos-polypeptide, predicted molecular weight includingphorylation, which affects substrate site availabilitytag Å 101,920 Da, which could be detected by both[7,35]. The cGMP-binding domain also appears to becoomassie staining and immunoblotting (Fig. 4A).coincident with the dimerization domain of PDE5 [7].Furthermore, lysate from PDE5A1 infected cells re-The exact nature of these molecular interactions and

their physiological significance require further study, sulted in a 76-fold higher level of cGMP hydrolyzing

FIG. 4. Expression and activity analysis of recombinant human PDE5A1. Extracts of PDE5A1 infected (PDE5A1) and mock infected(control) Sf9 cells were analyzed: A: PAGE followed by coomassie staining and western blotting. Full length PDE5A1 (arrow), predictedmolecular weight Å 101,861 Da, and size markers are indicated left of panel. B: PDE activity as determined by measuring cGMP hydrolysis.

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molecular cloning and expression of human cgmp-binding cgmp-specific phosphodiesterase (pde5)

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