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• W2030002022 abstract "We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCα and PfGCβ) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCβ inEscherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCα was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct. We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCα and PfGCβ) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCβ inEscherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCα was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct. adenylyl cyclase guanylyl cyclase cAMP-dependent protein kinase polymerase chain reaction base pair(s) open reading frame reverse transcription The life cycle of the human malaria parasite Plasmodium falciparum is complex with several stages that differ at both the morphological and biochemical levels. Mosquito transmitted sporozoites migrate to the liver where they undergo asexual multiplication within hepatocytes. Liver forms are then released into the bloodstream where they invade red blood cells. A small proportion of the erythrocytic forms develop into gametocytes, precursors of male and female gametes. When gametocytes are taken up by a mosquito during a bloodmeal, they must first emerge from the red blood cells as gametes before fertilization occurs in the midgut, prior to completion of the insect stages of the parasite life cycle. The emergence of eight motile male gametes from a red blood cell is known as exflagellation. Recent work (1.Bilker O. Lindo V. Panico M. Etienne A.E. Paxton T. Dell A. Rodgers M. Sinden R.E. Morris H.R. Nature. 1998; 392: 289-292Crossref PubMed Scopus (464) Google Scholar) has identified xanthurenic acid, a metabolite of tryptophan, as a putative in vivo gametocyte-activating factor. In bothP. falciparum and Plasmodium berghei there is evidence that cyclic GMP may be involved in the development and emergence of male gametes (2.Kawamoto F. Alejo-Blanco R. Fleck S.L. Kawamoto Y. Sinden R.E. Mol. Biochem. Parasitol. 1990; 42: 101-108Crossref PubMed Scopus (96) Google Scholar). The parasite signaling pathways involved in these processes remain to be elucidated. Two key components of signal transduction pathways are adenylyl cyclase (AC)1 and guanylyl cyclase (GC), enzymes that catalyze the conversion of ATP and GTP to cAMP and cGMP, respectively. Membrane-associated mammalian ACs are activated indirectly following interaction of a ligand with a separate receptor. This in turn binds to an intracellular heterotrimeric GTP-binding protein (G protein). Subunits of the activated G protein then interact with AC stimulating the synthesis of cAMP, which activates cAMP-dependent protein kinase (PKA). PKA phosphorylates a number of proteins; its final destination is often the nucleus where it activates transcription factors, thereby changing the pattern of gene expression. In mammals, nine distinct membrane-localized ACs have so far been characterized (3.Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (742) Google Scholar). They conform to the same basic structure comprising two sets of six hydrophobic transmembrane domains, each of which is followed by a cytoplasmic catalytic region termed C1 and C2, respectively. The amino-terminal portions (C1a and C2a) are homologous with each other and between species (4.Krupinsky J. Coussen F. Bakalyar H.A. Tang W.-J. Feinstein P.G. Orth K. Slaughter C. Reed R.R. Gilman A.G. Science. 1989; 244: 1558-1564Crossref PubMed Scopus (508) Google Scholar). Guanylyl cyclase exists in two main forms. The receptor form (with a single membrane-spanning domain) binds directly to an extracellular ligand leading to activation and production of intracellular cGMP. The soluble, cytosolic form of the enzyme is activated by nitric oxide in the presence of heme (reviewed in Ref. 5.Chinkers M. Garbers D.L. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar). GCs have a variety of roles in higher organisms, for example in photoreceptor signal transduction (reviewed in Ref. 6.Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar). GCs have also been identified in several lower organisms including protozoans such as Dictyostelium (7.Janssens P.M. de Jong C.C. Biochem. Biophys. Res. Commun. 1988; 150: 405-411Crossref PubMed Scopus (16) Google Scholar),Paramecium, and Tetrahymena (8.Schultz J.E. Klumpp S. Methods Enzymol. 1991; 195: 466-474Crossref PubMed Scopus (10) Google Scholar, 9.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). In sea urchins, a membrane-bound GC found on the surface of sperm can act as a receptor for peptides released by the eggs, which influence sperm chemotaxis (reviewed in Ref. 10.Ward G.E. Moy G.W. Vacquier V.D. Adv. Exp. Med. Biol. 1986; 207: 359-382PubMed Google Scholar). cGMP often exerts its function by binding to and activating cGMP-dependent protein kinase, which phosphorylates and regulates the activity of a number of specific proteins. As part of an investigation of the role of cyclic nucleotides in signal transduction in the malaria parasite, we have isolated two genes fromP. falciparum that encode integral membrane proteins. These proteins are unusual in that they appear to be bifunctional. The amino-terminal regions have strong similarity to P-type ATPases, and the carboxyl-terminal regions, including domains with GC activity, conform to a structure normally associated with G protein-dependent ACs. P. falciparum clones 3D7A (11.Walliker D. Quakyi I.A. Wellems T.E. McCutchan T.F. Szarfman A. London Corcoran L.M. Burkot T.R. Carter R. Science. 1987; 236: 1661-1666Crossref PubMed Scopus (577) Google Scholar), T996 (12.Thaithong S. Beal G.H. Fenton B. McBride J. Rosario V. Walker A. Walliker D. Trans. Roy. Soc. Trop. Med. Hyg. 1984; 78: 242-245Abstract Full Text PDF PubMed Scopus (131) Google Scholar), and strain K1 (12.Thaithong S. Beal G.H. Fenton B. McBride J. Rosario V. Walker A. Walliker D. Trans. Roy. Soc. Trop. Med. Hyg. 1984; 78: 242-245Abstract Full Text PDF PubMed Scopus (131) Google Scholar) were cultured in flasks with RPMI 1640 medium supplemented with 25 mm Hepes, 0.1 mmhypoxanthine, 10% human serum (A+) as described previously (13.Harte P.G. Rogers N.C. Targett G.A.T. Immunology. 1985; 56: 1-7PubMed Google Scholar). Cultures contained A+ human red blood cells at a 10% hematocrit. Large scale parasite preparations of clone 3D7A were produced using a semi-automated continuous flow apparatus. Gametocytes (stages III–V) were harvested and purified by Percoll gradient centrifugation (13.Harte P.G. Rogers N.C. Targett G.A.T. Immunology. 1985; 56: 1-7PubMed Google Scholar). Genomic DNA was isolated from 109 mixed asexual erythrocytic stage parasites according to standard procedures (14.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Briefly, the parasites were first released from red blood cells by treatment with 0.1% saponin in phosphate-buffered saline. The cell pellet was then washed twice with phosphate-buffered saline and incubated in a solution containing 100 μg ml−1 proteinase K, 0.5% SDS, 100 mmNaCl, 10 mm Tris, pH 8.0, 25 mm EDTA at 37 °C for 16 h. DNA was further purified by phenol extraction and ethanol precipitation. Total RNA was isolated from 5 × 108 gametocytes (mainly stages III–V) or mixed asexual erythrocytic stage parasites by lysis with 4 m guanidine thiocyanate and CsCl centrifugation (15.Robson K.J.H. Jennings M.W. Mol. Biochem. Parasitol. 1991; 46: 19-34Crossref PubMed Scopus (70) Google Scholar). A 220-bp fragment of the PfGCα gene was isolated from genomic DNA (strain K1) by PCR using the degenerate primers DC1 (sense) and MS2 (antisense), which were derived from conserved regions of AC/GC genes from diverse species. The reactions (50 μl) were carried out in 10 mm Tris, pH 8.8, 50 mm KCl, 0.5–5 mm MgCl2, and 1% Triton X-100 with 1 μm of each primer, 200 μm dNTP mix, 1 unit of Biotaq enzyme (Bioline) and 1–50 ng of template DNA. To minimize nonspecific amplification products, the “touchdown” method (16.Don R.H. Cox P.T. Wainwright B.J. Baker K. Mattick J.S. Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2245) Google Scholar) of thermal cycling was used with the following conditions: first cycle, 94 °C (2.5 min), 41 °C (1 min), and 72 °C (1 min); and second cycle, 94 °C (45 s), 40 °C (1 min), and 72 °C (1 min). The annealing temperature was then decreased by 1 °C each two cycles until it reached 35 °C. 25 cycles were performed under these conditions. A final cycle was then carried out at 94 °C (45 s), 35 °C (1 min), and 72 °C (10 min). Inverse PCR (17.Triglia T. Peterson M.G. Kemp D.J. Nucleic Acids Res. 1988; 16: 8186Crossref PubMed Scopus (724) Google Scholar) was used to extend the available PfGCα sequence after library screening (below) failed to isolate the full-length gene. Based on Southern blot data, an AluI digest of genomic DNA (1 μg, strain K1) was circularized with T4 DNA ligase, and an overlapping fragment was obtained by inverse PCR using primers IPCR1 and IPCR2. A λGEM-12 library was constructed with P. falciparum (strain K1) genomic DNA using a Promega kit according to the manufacturer's instructions. Briefly, a Sau3a partial digest of genomic DNA was end-filled and ligated with blunt-ended XhoI cut λGEM-12 arms. After ligation, the DNA was packaged in vitro using a Packagene Extract (Promega) and plated on Escherichia colistrain LE392. The library (containing 2.7 × 105clones) was screened with the original 220-bp PCR product using standard procedures (14.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and a genomic clone (λ1) containing part of the PfGCα gene was isolated. Vectorette libraries were constructed as described previously (18.Li J.-L. Robson K.J.H. Targett G.A.T. Baker D.A. Eur. J. Biochem. 1996; 241: 805-813Crossref PubMed Scopus (73) Google Scholar) using 1 μg of genomic DNA (clone 3D7A) that had been digested with one of several restriction enzymes. This DNA (200 ng) was ligated into blunt-ended Vectorette DNA (Cambridge Research Biochemicals). Based on known sequence and Southern blot information, PCR was performed using specific primers (AC1–AC9 forPfGCα, pvec1–3 for PfGCβ) and the Vectorette I primer (Cambridge Research Biochemicals). With this procedure we were able to obtain the remainder of the PfGCα coding sequence and the cyclase-encoding domain of PfGCβ. In the PCR reactions, the polymerase was added to the tubes in the cycling block when the reaction temperature was at least 80 °C. This “hot start” and the use of annealing temperatures in the range 60–70 °C were important for the success of the reaction. The following oligonucleotides were used: DC1, 5′-GTATATAAAGTAGAAAC(A/T)AT(T/A)GG; MS2, 5′-(T/A)CC(A/G)AA(T/G)AA(A/G)CA(A/G)TA(T/A)C(T/G)(T/A)GGCAT; IPCR1, 5′-TTATGATCATCAATAAATAC; IPCR2, 5′-TTATTATTCATATCAGC; AC1, 5′-TGTATCACCATCGATTGTATTAAATG; AC2, 5′-CATAATAATTACTTGTATCACCATCG; AC3, 5′-ACGAGTAGGTTCAATACTGGCCAC; AC4, 5′-GGAAATAAAACTGCTTGAAATACTCC; AC5, 5′-GAATATGCTTCTGTTTAAATTCCTTTTTG; AC6, 5′-TTTTGAATATGCTTCTGTTTAAATTCC; AC7, 5′-GGCATTACTATTTTCTAAGGCAGC; AC8, 5′-ATGAGCAAGCATCATCATACATTC; AC9, 5′-TCTTCTTTGTCTTCATATGCATAGG; 5CAT, 5′-CGCGCGAATTCTCTACCTATTGTAAAGAATCA; 3CAT, 5′-CGCGCGAATTCCAAATTCGACTGGCGATCGTT; pvec1, 5′-ATCAATTCGTTTACTATATCTACTATC; pvec2, 5′-TTAACAGATCCAATCACACCACTG; and pvec3, 5′-ACGTGTGGTTGTAATGTAGATACC (degenerate positions are marked with slashes). To isolate chromosome-sized DNA, asexual blood stage parasites were first released from red blood cells by saponin lysis and then washed in TSE buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 50 mm EDTA). The pellet was resuspended in 9 volumes of TSE and then mixed with an equal volume of 1.6% agarose in TSE and pipetted into a gel mold (Bio-Rad). The agarose blocks (108-109 parasites/block) were incubated in 1% sarcosinate, 0.5 m EDTA, pH 8.0, 2 mg ml−1 proteinase K for 48 h at 50 °C and then stored at 4 °C. Chromosome separations were performed with 1% agarose gels using a contour-clamped homogeneous field electrophoresis system (CHEF DRII, Bio-Rad). The gels were run in 0.5% TBE buffer at 100 V for 96 h with a pulse time of 360 s ramped to 800 s. Typically, hybridizations were performed at 42 °C in 6× SSC and 50% formamide with washes in 1× SSC at 50 °C. For lower stringency experiments, hybridizations were at 50 °C in 5× SSC with washes in 2× SSC at room temperature. Northern blots were carried out using standard conditions (15.Robson K.J.H. Jennings M.W. Mol. Biochem. Parasitol. 1991; 46: 19-34Crossref PubMed Scopus (70) Google Scholar). Oligonucleotide primers 5CAT and 3CAT were designed to amplify the carboxyl-terminal region of PfGCα, Ser3914–Leu4226), incorporating the C2 catalytic domain. EcoRI restriction sites were included in the primers to facilitate insertion of the fragment in-frame with theSchistosoma japonicum glutathione S-transferase gene of the pGEX1λT plasmid (Amersham Pharmacia Biotech). Expression of the fusion protein was induced in a 500-ml mid-log phase E. coli (strain JM109) culture (A 600 = 0.4–0.5) by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm for 2 h. Cells were resuspended in phosphate-buffered saline /1% Triton X-100 and sonicated on ice. The soluble fusion protein was bound to 2 ml of glutathione-agarose (Sigma) on a Poly-Prep column (Bio-Rad) according to the manufacturer's instructions. Bound protein was eluted by addition of 5 mm reduced glutathione and dissolved in 0.5 ml of normal saline at a final concentration of 2 mg ml−1. This was mixed with 0.5 ml of Ribi Adjuvant System (Sigma) resuspended in 1 ml of normal saline. The mixture was homogenized by vortexing, and a New Zealand White rabbit was immunized at six intradermal sites (50 μg each), two intramuscular sites (200 μg each), and two subcutaneous sites (100 μg each). Similar booster injections were given at 3–4-week intervals. Preimmune serum was taken prior to immunization. Percoll-purified gametocytes that had been stimulated to undergo gametogenesis were fixed for 30 min with 2% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline, dehydrated in ethanol, embedded in LR White (London Resin Co.), and polymerized at 50 °C for 48 h. Ultrathin sections were collected on piloform-coated nickel grids and incubated with primary antibody and gold-conjugated secondary antibody as described previously (19.Baker D.A. Daramola O.O. McCrossan M.V. Harmer J. Targett G.A.T. Parasitology. 1994; 108: 129-137Crossref PubMed Scopus (46) Google Scholar). The sections were silver enhanced using an Amersham Pharmacia Biotech IntenSE M silver enhancement kit and then stained with a saturated uranyl acetate solution in 30% methanol followed by Reynolds lead citrate solution. The samples were analyzed with a JEOL 1200EX transmission electron microscope at 80 kV and photographed using Agfa Scienta EM film. DNA sequencing was performed using an ABI PRISM 377 DNA sequencer. The reactions were performed with a dye terminator cycle sequencing Ready Reaction Kit (Perkin-Elmer). Preliminary sequence data for P. falciparum chromosome 11 were obtained from the Institute for Genomic Research website. Sequencing of chromosome 11 was part of the International Malaria Genome Sequencing Project and was supported by an award from the NIAID, National Institutes of Health. Sequence data for P. falciparum chromosome 13 were obtained from the Sanger Center website. Sequencing of P. falciparum chromosome 13 was accomplished as part of the Malaria Genome Project with support by the Wellcome Trust. The cyclase catalytic domains of both PfGCα (C1, Gly2998–Val3329, and C2, Gln3956–Lys4163) and PfGCβ (C1, Val1533–Tyr1758, and C2, Glu2946–Ala3122) and the catalytic domain of aTrypanosoma cruzi AC (ADC-1, Arg849–Lys1169) were cloned into pTrcHis C (Invitrogen) to give hexahistidine amino-terminal fusion proteins. Expression of the fusion proteins in E. coli (strain TP610, AC-deficient) was induced at 30 °C by incubation with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 1 h. Cultures were then lysed by sonication (six 10-s bursts at an amplitude of 20 μm using an MSE Soniprep 150) in a solution containing 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 5 mm imidazole, 0.1% Triton X-100, 10 mm β-mercaptoethanol, 1 mmphenylmethylsulfonyl fluoride, 50 μg ml−11-chloro-3-tosylamido-7-amino-2-heptanone, and 1 mg ml−1lysozyme). Lysates were cleared by centrifugation at 15,000 ×g for 20 min at 4 °C. Proteins were affinity purified on Ni2+-nitrilotriacetic acid Sepharose according to the manufacturer's instructions (Qiagen). AC and GC activity was assayed in reaction volumes of 40 μl. 30-μl aliquots of fusion protein (2–5 μg) were added to 10 μl of 4× reaction buffer (40 mm Tris, pH 8.0, containing either ATP or GTP at 4 mm and 8 mm MgCl2 or MnCl2) and incubated at 30 °C. Reactions were stopped by addition of 10 μl of 0.2 m EDTA, pH 8.0, followed by boiling for 2 min. For the t = 0 time points, EDTA was added before the protein. cAMP or cGMP concentrations were measured in 96-well plates using a competition assay (20.Gilman A.G. Adv. Cyclic Nucleotide Res. 1972; 2: 9-24PubMed Google Scholar) with purified regulatory subunit of beef heart cAMP-dependent protein kinase as the cAMP-binding protein (PKA-R) or highly specific anti-cGMP antibodies as the cGMP binding protein (21.Schulkes C.C.G.M. Schoen C.D. Arents J.C. Van Driel R. Biochim. Biophys. Acta. 1992; 1135: 73-78Crossref PubMed Scopus (16) Google Scholar). Briefly, samples were mixed with 20 μl of 0.4 mCi of [3H]cAMP (48 Ci mmol−1; Amersham Pharmacia Biotech) or [3H]cGMP (14.8 Ci mmol−1) per ml of assay buffer (4 mm EDTA in 150 mm sodium phosphate buffer, pH 7.5) and 20 μl of PKA-R or cGMP antibody preparation. After incubation on ice for 90 min, 40 μl of activated charcoal (1.25 g of charcoal and 0.5 g of bovine serum albumin in 25 ml of assay buffer) was added to adsorb unbound [3H]cAMP or [3H]cGMP. Samples were centrifuged at 3,000 × g for 15 min at 4 °C. 50 μl of supernatant was mixed with 100 μl of scintillation fluid, and the radioactivity was measured (22.van Es S. Virdy K.J. Pitt G.S. Meima M. Sands T.W. Devreotes P.N. Cotter D.A. Schaap P. J. Biol. Chem. 1996; 271: 23623-23625Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A standard curve was produced with a linear range from 0–16 pmol cAMP/cGMP. Degenerate oligonucleotide primers were designed based on conserved regions in the catalytic domains of AC/GC from other species (see “Experimental Procedures”). Using these, a 220-bp fragment was amplified by PCR from P. falciparum genomic DNA. Data base searches suggested that the product was derived from a purine nucleotide cyclase gene homologue. This fragment was used to screen a genomic DNA library, and a clone was isolated. Sequence analysis of a 5.5-kilobaseBamHI-EcoRV fragment identified a 1.8-kilobase open reading frame (ORF) corresponding to the 3′-end of a cyclase-like gene, with a putative stop codon and adjacent 3′ noncoding sequence. Inverse and Vectorette PCR techniques (see “Experimental Procedures”) were used to extend the sequence toward the 5′-end of the gene. This generated an uninterrupted ORF of approximately 10 kilobases. A putative start codon was then identified following searches of the Institute for Genomic Research P. falciparumdata base. Verification of the gene structure was obtained by sequencing the corresponding region (Fig.1), amplified from genomic DNA. In addition the entire sequence of PfGCα has been confirmed following release of data from the Institute for Genomic Research data base, which became available during the preparation of this manuscript. The nucleotide sequence of PfGCα comprises a single ORF of 12,678 bp (these sequence data have been submitted to the GenBankTM data base under accession number AJ245435). The A/T content (75.6%) and codon usage conform to that of all P. falciparum genes reported to date. The designated termination codon (TAA) of PfGCα occurs in a position analogous to that in mammalian-type AC (4.Krupinsky J. Coussen F. Bakalyar H.A. Tang W.-J. Feinstein P.G. Orth K. Slaughter C. Reed R.R. Gilman A.G. Science. 1989; 244: 1558-1564Crossref PubMed Scopus (508) Google Scholar, 23.Bakalyar H.A. Reed R.R. Science. 1990; 250: 1403-1406Crossref PubMed Scopus (525) Google Scholar, 24.Defer N. Marinx O. Stengel D. Danisova A. Iourgenko V. Matsuoka I. Caput D. Hanoune J. FEBS Lett. 1994; 351: 109-113Crossref PubMed Scopus (33) Google Scholar, 25.Zhang G. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (325) Google Scholar) and is followed immediately by an extremely A/T-rich region (95%). Within the ORF, two regions of unusually high A/T content were identified (nucleotides 7350–7530 and nucleotides 9060–9450). To investigate the possibility of introns at these positions and elsewhere in the gene, a series of RT/PCR reactions were carried out. There were no differences in the sizes of the products obtained from RNA (DNase I-treated) and genomic DNA (data not shown). In negative controls where the reaction was performed in the absence of RT, no products were amplified. Using this approach it was shown that PfGCα was uninterrupted by introns. Analysis of the PfGCα sequence revealed an unexpected finding; the gene had the potential to encode a bifunctional protein in which the amino-terminal domain had high similarity to P-type ATPases (26.Ripmaster T.L. Vaughn G.P. Woodford Jr., J.L. Mol. Cell. Biol. 1993; 13: 7901-7912Crossref PubMed Scopus (96) Google Scholar), and the carboxyl terminus was a structural homologue of G protein-dependent adenylyl cyclases. The nucleotide sequence adjacent to the putative start codon is aaaaATG, which is similar to the consensus sequence upstream of many P. falciparum genes (27.Saul A. Battistutta D. Mol. Biochem. Parasitol. 1990; 42: 55-62Crossref PubMed Scopus (50) Google Scholar). The sequence immediately upstream of this is preceded by a highly A/T-rich sequence interrupted by stop codons in all three reading frames. A P. falciparum λGEM-12 library was screened at low stringency with a probe corresponding to the PfGCαC2 catalytic domain (see “Experimental Procedures”). A clone containing 1.3 kilobase of sequence with similarity to PfGCα and encompassing an in-frame stop codon was isolated. This gene was designated PfGCβ. The remainder of the cyclase encoding region (an additional 2000 nucleotides) was obtained from Vectorette libraries (see “Experimental Procedures”). Upstream, an additional open reading frame with a high degree of similarity to P-type ATPases was discovered. The sequence of this region was obtained from the P. falciparum genome project (Sanger Center). The cyclase and the ATPase coding regions were found to be separated by 241 bp of A/T-rich sequence. This was shown to be an intron, and both coding regions were found to form a contiguous ORF. Sequence analysis of a series of RT/PCR products obtained using primers specific to both the cyclase and ATPase domains demonstrated the presence of 12 introns in thePfGCβ sequence. These were all confined to the ATPase encoding domain (Fig. 1). The fully spliced transcript has the potential to encode a bifunctional protein of 3122 amino acids (accession number AJ249165). The 5′-most exon contains an A/T-rich sequence upstream of the designated start codon that has stop codons in all three reading frames. Sequencing of RT/PCR products has confirmed that this A/T-rich sequence forms part of the mature mRNA. The predicted stop codon (position 9367) is located upstream of a region with an extremely A/T-rich composition that has stop codons in all three reading frames. The positions of the designated start and stop codons are consistent with those found in other genes encoding similar P-type ATPases and cyclases, respectively. The sequence of the cyclase-encoding region has been confirmed by chromosome 13 sequence data that were released (Sanger Center) during preparation of this manuscript. Using the Genestream Align global alignment program the sequences of PfGCα and PfGCβ were found to share 22% identity in their cyclase domains and 19% in their ATPase domains. The relatedness of both PfGCα and PfGCβ to GC/AC is concentrated in two regions (PfGCαC1 Gly2999–Val3329; PfCαC2, Gln3956–Lys4163; PfGCβC1, Val1533–Tyr1758; and PfGCβC2, Glu2946–Ala3122), which have 26–33% sequence identity and 48–52% similarity (28.Altschul S.F. Gish W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70715) Google Scholar) to the corresponding regions of enzymes from diverse species. These hydrophilic regions correspond to the positions of the catalytic domains. PfGCαC1 is characterized by the presence of a long asparagine-rich stretch (amino acids Asn3082–Asn3138; Fig.2). Interestingly, the aggregation-specific adenylyl cyclase (ACA) of the protozoanDictyostelium discoideum has a similar sequence insertion at the same position within the C1 catalytic domain (29.Pitt G.S. Milona N. Borleis J. Lin K.C. Reed R.R. Devreotes P.N. Cell. 1992; 69: 305-315Abstract Full Text PDF PubMed Scopus (264) Google Scholar). There is a second, shorter insertion in PfGCαC1(Tyr3186–Thr3225; Fig. 2) that corresponds (in terms of both position and size) to an insert found in all of the trypanosomatid ACs sequenced to date. No such inserts are present in the cyclase catalytic domains of PfGCβ. Alignment of PfGCαC1 with PfGCαC2 revealed 23% sequence identity, whereas alignment of PfGCβC1 and PfGCβC2 gave 34% identity. The cyclase catalytic domains of both PfGCα and PfGCβ are each preceded by highly hydrophobic regions that in membrane-associated mammalian ACs have been predicted to form two sets of six transmembrane domains. The PfGCα and PfGCβ sequences are also compatible with this type of organization (Fig. 3). The putative ATPase domains of both proteins conform to the P-type (or E1-E2) family of cation transporting ATPases (30.Jencks W.P. Ann. N. Y. Acad. Sci. 1992; 671: 49-56Crossref PubMed Scopus (19) Google Scholar). The model for this family of enzymes predicts 10 transmembrane domains arranged as a set of four toward the amino terminus and a set of six near the carboxyl terminus. The amino acid sequences of both PfGCα and PfGCβ are consistent with this type of structure (Fig. 3). For both PfGCα and PfGCβ the relatedness to P-type ATPases from diverse species is concentrated in two regions and is summarized below. The amino-terminal domain of PfGCα has 26–32% identity and 48–52% similarity over 250–300 amino acids; for the carboxyl terminus the figures are 28–30% identity and 48–50% similarity. For PfGCβ, the amino-terminal domain has 23–25% identity and 43–46% similarity; for the carboxyl terminus the figures are 21–23% identity and 46–48% similarity. The membrane-spanning regions of the P-type ATPase domains are more highly conserved than are those of the cyclase domains. This might be expected given the potential functional significance of these transmembrane regions. The consensus phosphorylation site of P-type ATPases (DKTGTLT) is present in PfGCα (Asp756–Thr762) but not in PfGCβ. This signature sequence is involved in formation of an aspartyl phosphate intermediate during ATP hydrolysis (31.MacLennan D.H. Brandl C.J. Korczak B. Green N.M. Nature. 1985; 316: 696-700Crossref PubMed Scopus (804" @default.
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