Molecular Cloning and Expression of Human cGMP-Binding cGMP-Specific Phosphodiesterase (PDE5)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

247, 249–254 (1998)

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Molecular Cloning and Expression of Human cGMP-Binding cGMP-Specific Phosphodiesterase (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

A human PDE5 cDNA has been isolated which contains an open reading frame encoding an 875 amino acid, 100,012 Da polypeptide, the expression of which yields a protein of the predicted size and is capable of hydrolyzing cGMP. The deduced amino acid sequence is very similar (95%) to that of bovine PDE5, and comprises a conserved cGMP-binding domain and catalytic domain. Northern analysis reveals a major and minor transcript of Ç9 kb and Ç8 kb respectively, thus indicating the existence of at least two splice variants, the major form being readily detected in bladder, colon, lung, pancreas, placenta, prostate, small intestine, and stomach. q 1998 Academic Press

The cyclic nucleotides cAMP and cGMP function as second messengers for a plethora of extracellular signalling molecules, such as neurotransmitters and hormones, and mediate their effects by interaction with a variety of intracellular targets, e.g., kinases, ion channels and transcription factors. Their intracellular levels are regulated through a dynamic balance of the rates of synthesis by cyclases and degradation by cyclic nucleotide phosphodiesterases (PDEs). PDEs form a superfamily of enzymes which catalyse the hydrolysis of 3*:5*-cyclic nucleotides to the corresponding nucleoside 5*-monophosphates. On the basis of their substrate specificities, kinetic properties, regulatory features and AA sequences, the various PDEs 1

The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with Accession Number AJ004865. 2 Corresponding author. Fax: /44 (0)1304 615600. E-mail: [email protected]. Abbreviations: AA, amino acid; bp, base pair; Da, Dalton; EDTA, ethylene diamine tetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-N*-(2-ethanesulfonate); PAGE, polyacrlyamide gel electrophoresis; PCR, polymerase chain reaction; PDE, phosphodiesterase; SSC, saline sodium citrate buffer; SDS, sodium dodecyl sulfate

are currently subdivided into 7 major families [1,2]. Each family, and even members within a family, exhibit distinct tissue, cell and subcellular expression patterns and hence participate in discrete signal transduction pathways [1]. The PDE5 (cGMP-binding cGMP-specific PDE) family is of particular interest due to its involvement in the NO/cGMP signaling pathway, which modulates smooth muscle tone [3], and the development of sildenafil (VIAGRAy, UK-92,480), an orally active PDE5 inhibitor. Sildenafil is efficacious in the treatment of male erectile dysfunction [4] by potentiating NO mediated increases in cGMP in corpus cavernosal smooth muscle [5]. PDE5 was first purified and characterized from rat [6] and bovine [7] lung, with enzyme activity also being shown to be present in a variety of other tissues including platelets, spleen, and vascular smooth muscle [8,9,10]. Bovine PDE5 is specific for cGMP hydrolysis with the native protein having a moderate affinity for cGMP (Km Å 5.6 mM) and no significant hyhrolytic activity against cAMP [7]. Protein sequencing and molecular cloning subsequently lead to the isolation of a PDE5 cDNA from bovine lung which encoded a protein of 865 AA residues [11] - denoted as BTPDE5A1 in accordance with standardized nomenclature [2]. More recently, a rat PDE5 cDNA has been described which appears to be a distinct splice variant, i.e., PDE5A2 [12]. PDE5 comprises a C-terminal catalytic domain of Ç250 AA residues, the sequence of which is well conserved between all mammalian PDEs [13], and an Nterminal cGMP-binding domain; this is supported by the finding that proteolytic digestion of bovine PDE5 liberates two distinct domains [7]. The cGMP-binding domain of PDE5 spans Ç380 AA residues and is also a conserved feature of the PDE6 (photoreceptor PDE) and PDE2 (cGMP-stimulated PDE) families [14]. This domain constitutes a distinct cGMP-binding element which bears no significant sequence homology to that of other cGMP-binding proteins, e.g., the catabolite gene activator protein family [15,16], nor the guanine nucleotide binding domain of G-proteins [17].

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We report here the cDNA cloning of a functionally active human PDE5, HSPDE5A1, and an analysis of its deduced AA sequence and its expression in a range of tissues. MATERIALS AND METHODS PCR Isolation of a Partial Human PDE5 cDNA

on both strands, the encoded AA sequence being: MDYKDDDDKGSRSEARGIQRRAG-human PDE5A1 (Fig. 1; FLAG tag and linkerderived sequence underlined). Recombinant viral stocks were prepared using the Bac-toBac system (Life Technologies) according to the manufacturer’s protocol, and Sf9 cells were cultured in Sf 900 II serum-free media (Life Technologies) at 277C. For expression, 1 1 108 cells in 100 ml were infected at a multiplicity of infection of 1. Cells were harvested 48 hours post-infection for assay.

Northern Blotting Analysis

A sense (5*-GGCATCGTGGGMCAYGTSGCM-3*) and antisense primer (5*-GACAGCTCAAAGTCRCTGAAGYKRAA-3*) corresponding to the cGMP-binding and catalytic regions respectively were used. PCR reactions contained Ç10 ng human lung cDNA (Clontech, Palo Alto, CA), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2 , 0.2 mM dNTPs, 2.5 units of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT) and each primer at 300 nM. Two rounds of amplification were carried out: 957C/1.5 min, 507C/1.5 min, 727C/3 min, 34 cycles; 957C/1.5 min, 507C/1.5 min, 727C/7 min, 1 cycle. PCR products were subcloned into pUC18 using the Sureclone Ligation Kit (Pharmacia Biotech, Uppsala, Sweden), and plasmid DNA prepared (Qiagen, East Sussex, UK). All DNA sequencing was performed on both strands by fluorescence-tagged dye terminator cycle sequencing (Perkin-Elmer) and analyzed on an ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA).

cDNA Library Screening Approximately 1 1 106 plaque forming units from both a human prostate and human skeletal muscle lgt10 cDNA library (Clontech) were screened by hybridization using standard procedures [18]. The hybridization probe was generated by labeling the human PDE5 partial cDNA with [a-32P]dCTP using a Megaprime kit (Amersham, Bucks, UK) and purifying the reaction products with Chromaspin30 columns (Clontech). Purified, positive l phage were analyzed by sequencing, i.e., phage were used as a template in PCR reactions containing a lgt10-forward primer (5*-CGAGCTGCTCTATAGACTGC-3*) and a lgt10-reverse primer (5*-GGGTAAATAACAGAGGTGGC -3*) at 1 mM with 30 rounds of amplification: 947C/10 min; 557C/1 min, 727C/4 min, 947C/30 sec, 30 cycles; 557C/1 min, 727C/10 min, 1 cycle. Each PCR product was directly sequenced from both ends using nested lgt10 primers (5*-ATGAGTATTTCTTCCAGGGT3* and 5*-TGAGCAAGTTCAGCCTGGTT-3*). Selected clones were further characterized by preparing phage DNA [18] and subcloning the inserts into pBluescript KS/ (Stratagene, La Jolla, CA) for sequencing and subsequent subcloning steps. The sequence of the clones used to derive a consensus for PDE5A1 was determined on both strands.

Subcloning and Expression of Human PDE5A1

A HindIII/PstI fragment encoding the catalytic domain of human PDE5A was labeled with [a-32P]dCTP using a Megaprime kit (Amersham), and reaction products (probe) purified using Chromaspin-30 columns (Clontech). Multiple Tissue Northern blots were purchased (Clontech), prehybridized in ExpressHyb (Clontech) at 687C for 6 hours and hybridized (Ç1 1 106 cpm probe/ml) at 687C for 16 hours. Blots were washed at 3 1 SSC, 1% (w/v) SDS at 457C followed by 1 1 SSC, 1% (w/v) SDS at 457C and exposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA) overnight. Blots were checked for equal loading of poly(A)/ RNA in each lane using a human b-actin cDNA probe as described above (data not shown).

Western Analysis PDE5A1 infected and mock infected cell lysates (Ç1 1 104 cell equivalents) were separated by denaturing PAGE using the NuPAGE mini-gel system (Novex, San Diego, CA) and either stained with coomassie or transferred to a polyvinylidene difluoride membrane (Novex) for immunoblotting. Western analysis was performed by enhanced chemiluminescence (Amersham) according to the manufacturer’s protocol, using an anti-FLAG antibody (Sigma, Dorset, UK) and a horse radish peroxidase conjugated anti-mouse IgG (BioRad, Herts, UK) as a secondary antibody at 1:500 and 1:750 dilutions respectively.

Enzyme Preparation and Phosphodiesterase Assay Transfected Sf9 cells were harvested by centrifugation (1,000 1 g for 10 min), resuspended in homogenization buffer (20 mM HEPES pH 7.2, 1 mM EDTA, 20 mM sucrose, 150 mM NaCl and 1 protease inhibitor tablet (Boehringer) per 50 ml) at 1 1 107 cells/ml and disrupted by sonication. Cellular debris was removed by centrifugation at 14,000 1 g for 10 min and supernatant stored in aliquots at 0707C. PDE activity was measured using a modification of the method of Hurwitz et al. [20], i.e., in assay buffer containing 40 mM Tris-HCl pH 7.4, 10 mM MgCl2 and 2 mg/ml bovine serum albumin (final concentrations) with 0.5 mM [3H]cGMP (Amersham) as substrate in a final volume of 100 ml. All assays were performed in triplicate.

RESULTS AND DISCUSSION

A composite clone which encoded the full length protein with an N-terminal epitope tag was constructed from 2 clones (Fig. 1) for expression in insect cells. The C-terminal half was isolated by PCR using a sense primer (5*-GCGAATTCAAGCTTTTGTCATCTTTTGTGGC-3*) covering a unique HindIII restriction site (Fig. 1) and an antisense primer (5*-GCTCTAGATTATTAGTTCCGCTTGGCCTGGCCGCTTTCC-3*) at the stop codon, the latter incorporated a tandem stop codon and unique XbaI restriction site to facilitate subcloning. PCR was performed using the Expand High Fidelity PCR system (Boehringer Mannheim, West Sussex, UK) and the following cycle conditions: 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 first two AA residues, was isolated as a BsrBI/HindIII fragment (Fig. 1) and ligated with the HindIII/XbaI fragment into the StuI/ XbaI sites of the baculovirus transfer vector pFASTBAC (Life Technologies, Gaithersburg, MD) which had been modified to include a 5* FLAG epitope tag [19]. The sequence of the insert was determined

A CLUSTAL alignment [21] of bovine PDE5 with members of the PDE6 family revealed 2 conserved regions around the cGMP-binding and catalytic domains, GIVGHVAA and FSFSDFELS respectively, which were used to design 2 partially degenerate PCR primers. These primers (Fig. 1) were used to isolate a partial human PDE5 cDNA from lung, the sequence of which exhibited 94% identity over the entire 1,020 bp to the corresponding sequence of bovine PDE5 [11] (data not shown). The partial human PDE5 cDNA was then used as a hybridization probe to screen human prostate and skeletal muscle cDNA libraries. This resulted in the 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 by two cDNA clones which span residues 1-2137 and 364-3041 (arrowed brackets), and the AA sequence of the catalytic domain is highlighted in bold text. The PCR primers used to isolate the partial human PDE5 cDNA are indicated (arrows), along with the restriction enzyme sites 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 were used for subsequent subcloning for expression. Sequencing of the human PDE5 cDNA clones lead to the assembly of a consensus nucleotide sequence (Fig. 1) and the identification of a 906 AA long open reading frame which could extend further in the 5* direction. However, an alignment of the deduced AA sequence with that of bovine PDE5 (Fig. 2.) suggests that the initiation codon is in fact the first methionine encountered (nucleotide position 94; Fig. 1). This is further supported by the occurrence of an identical match to the consensus Kozak sequence at this position, i.e., ACCATGG [22]. In addition, the predicted coding sequence of 875 AA (Fig. 1) would yield a 100,012 Da polypeptide which is in close agreement with the known molecular weight of native bovine PDE5 [7]. Therefore, we conclude that the human cDNA described here encodes an 875 AA, 100,012 Da polypep-

tide. Given the high degree of similarity (95%) to the AA sequence of bovine PDE5 (Fig. 2), even at the Nand C-termini, we propose that this cDNA encodes the human homologue hence is denoted as HSPDE5A1 in accordance with standardized nomenclature [2]. A BLAST2 alignment [23] of human PDE5A1 with the other known PDE5 AA sequences, i.e., bovine [11] and rat [12], reveals a high degree of conservation, 95% and 93% respectively, except at the N-terminus where the rat sequence differs significantly (Fig. 2). Presumably, the latter represents a distinct splice variant for which a human equivalent may exist. The functional significance of PDE splice variants is now beginning to be examined with evidence suggesting potential roles in regulating enzyme stability and, perhaps more crucially, both the activity of the enzyme and its location in the cell by conferring an ability to associate with specific cellular membranes [24].

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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 domain residues conserved between all known mammalian PDE families (bold, underlined asterisks) highlighted.

At the C-terminus of human PDE5A1 is a catalytic domain of Ç250 AA residues, this sequence being well conserved between all mammalian PDEs [13], which contains the PDEase signature motif HDX2HX4N [25]. The degree of similarity across this region for bovine and rat PDE5 is 97% and 95% respectively, followed most closely by human cone PDE6 (44%), which supports the assignment of this human PDE as PDE5A. Within this domain are 21 AA residues which are absolutely conserved between all known mammalian PDE families (Fig. 2) [26], therefore, they almost certainly play an important role in cyclic nucleotide binding and/ or hydrolysis. Interestingly, it has been suggested that the HX3HX24E motif which exists in tandem in mammalian PDEs, including human PDE5A1, functions to coordinate Zn2/ in the catalytic site similar to that of Zn2/ hydrolases such as thermolysin [27]. This accounts for 5 of the 21 AA residues; the glutamic acid residue at the end of the first repeat is either substituted for by aspartic acid or there is a nearby glutamic acid in PDEs where this motif is not conserved. However, the precise function of this motif and that of the remaining residues awaits further study. Like other cGMP-binding PDEs, human PDE5A1 comprises an N-terminal cGMP-binding domain which

spans Ç380 AA residues (Fig. 2). Overall this domain exhibits 97% and 93% similarity to that of bovine and rat PDE5 respectively, with human PDE6 having 27% similarity and human PDE2 26%. Within the cGMPbinding domain of human PDE5A1 are tandem repeats of the conserved sequence motif N(K/R)XnFX3DE [11] (Fig. 2), the NKXnD motif having been shown by mutagenesis to be important for cGMP binding [28]. For bovine PDE5 it has been demonstrated that the cGMP-binding domain contains two kinetically distinct sites, site a and site b, which have high and low dissociation constants for cGMP respectively [29] - Scatchard analysis suggests that this is also the case for PDE2 and PDE6 [30,31]. Furthermore, it has been shown that cGMP binding to PDE5 enhances phosphorylation at a single serine residue both in vitro [7] and in intact cells [32], and that both cGMP-binding sites need to be occupied for phosphorylation [33]. The phosphorylation site in bovine PDE5, serine-92, is also conserved in human PDE5A1 and rat PDE5 (Fig. 2), and is known to be phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases [7]. However, despite these studies, the functional significance of cGMP binding to this allosteric site and phosphorylation have yet to be elucidated since neither have an effect on the

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FIG. 3. Northern blotting analysis of PDE5A expression in human tissues. Approximately 2 mg poly(A)/ RNA per tissue (as indicated above lane) was probed with the catalytic domain of human PDE5A. Size markers are indicated left of panel.

kinetic properties of the enzyme [28,33]. It has been suggested that the cGMP-binding sites of PDE5 serve as a buffer to protect submicromolar levels of cGMP from hydrolysis [34] and that cGMP binding might induce a subtle conformational change, possibly via phosphorylation, which affects substrate site availability [7,35]. The cGMP-binding domain also appears to be coincident with the dimerization domain of PDE5 [7]. The exact nature of these molecular interactions and their physiological significance require further study,

possibly in intact cells or crude systems due to the possible involvement of additional regulatory factors. Analysis of the AA sequence of human PDE5A1 using PROSITE [36] did not reveal any additional protein motif that could be ascribed with confidence as being functionally significant. Northern analysis was performed on poly(A)/ RNA from a range of human tissues using the catalytic domain of human PDE5A1 as a probe. The data obtained indicate that PDE5A is expressed in the tissues examined as a major transcript of Ç9 kb and a less prominent transcript of Ç8 kb (Fig. 3), which suggests that at least two PDE5A splice variants exist. Given the coding region spans 2,625 bp, the 5* and/or 3* untranslated regions of the corresponding transcript are large and are not represented in the clones we isolated which resulted in a 3,041 bp composite cDNA (Fig. 1). Appreciable expression of PDE5A mRNA was observed in bladder, colon, lung, pancreas, placenta, prostate, small intestine and stomach, with low level expression in brain, heart, kidney, skeletal muscle and uterus and no detectable transcript in liver (Fig. 3) - low levels of PDE5 mRNA could result from blood vessels which are known to contain PDE5 activity [10]. These data are consistent with a role for PDE5 in smooth muscle. Finally, the ability of the human PDE5A1 cDNA to yield a functionally active protein of the predicted size was examined by expressing an epitope tagged construct in insect cells. The PDE5A1 expression construct resulted in the production of an Ç100 kDa polypeptide, predicted molecular weight including tag Å 101,920 Da, which could be detected by both coomassie staining and immunoblotting (Fig. 4A). Furthermore, lysate from PDE5A1 infected cells resulted 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), predicted molecular weight Å 101,861 Da, and size markers are indicated left of panel. B: PDE activity as determined by measuring cGMP hydrolysis. 253

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activity than mock infected (control) cell lysate (Fig. 4B). These data confirm that PDE5A1 is a bona fide cGMP-binding cGMP-specific PDE. The availability of the human PDE5A cDNA and recombinant human PDE5A will allow detailed biochemical analysis as well as permit characterization of the structure and function of the domains. This will improve our understanding of the mechanisms by which PDE5 activity is mediated and regulated, and hence its contribution to modulating cGMP-mediated signal transduction. REFERENCES 1. Beavo, J. A. (1995) Physiol. Rev. 75, 725–748. 2. Beavo, J. A., Conti, M., and Heaslip, R. J. (1994) Mol. Pharmacol. 46, 399–405. 3. Francis, S. H., and Corbin, J. D. (1994) in Cyclic GMP: Synthesis, Metabolism and Function (Murad, P., Ed.) Advances in Pharmacology Vol. 26, pp. 115–170, Academic, New York. 4. Boolell, M., Allen, M. J., Ballard, S. A., Gepi-Attee, S., Muirhead, G. J., Naylor, A. M., Osterloh, I. H., and Gingell, C. (1996) Int. J. Impot. Res. 8, 47–52. 5. Ballard, S. A., Gingell, C. J., Tang, K., Turner, L. A., Price, M. E., and Naylor, A. M. (1998) J. Urol. 159, in press. 6. Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722–729. 7. Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14964–14970. 8. Hamet, P., Coquil, J.-F., Bousseau-Lafortune, S., Franks, D. J., and Tremblay, J. (1984) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 16, 119–136. 9. Coquil, J.-F., Brunelle, G., and Guedon, J. (1985) Biochem. Biophys. Res. Commun. 127, 226–231. 10. Coquil, J.-F., Franks, D. J., Wells, J. N., Dupuis, M., and Hamet, P. (1980) Biochim. Biophys. Acta 631, 148–165. 11. McAllister-Lucas, L. M., Sonnenburg, W. K., Kadlecek, A., Seger, D. L., Trong, H. L., Colbran, J. L., Thomas, M. K., Walsh, K. A., Francis, S. H., Corbin, J. D., and Beavo, J. A. (1993) J. Biol. Chem. 268, 22863–22873. 12. Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K., and Omori, K. (1997) Eur. J. Biochem. 249, 434–442. 13. Charbonneau, H., Beier, N., Walsh, K. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9308–9312. 14. Charbonneau, H., Prusti, R. K., LeTrong, H., Sonnenburg, W. K., Mullaney, P. J., Walsh, K. A., and Beavo, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 288–292.

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