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• W2091879423 abstract "A cDNA (Schistosoma mansonitryptophan hydroxylase; SmTPH) encoding a protein homologous to tryptophan hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of serotonin, was cloned from the human parasite Schistosoma mansoni. Bacterial expression of SmTPH as a histidine fusion protein produced soluble active enzyme, which was purified to apparent homogeneity and a final specific activity of 0.17 μmol/min/mg of protein. The purified enzyme was found to be a tetramer of approximately 240 kDa with a subunit size of 58 kDa. Several of the biochemical and kinetic properties of SmTPH were similar to those of mammalian tryptophan hydroxylase. Unlike the mammalian enzyme, however, SmTPH was found to be stable at 37 °C, itst½ being nearly 23 times higher than that of a similarly expressed rabbit tryptophan hydroxylase. A semiquantitative reverse transcription polymerase chain reaction showed that the level of SmTPH mRNA in a larval stage of the parasite (cercaria) is 2.5 times higher than in adult S. mansoni, suggesting possible differences in the level of enzyme expression between the two developmental stages. This study demonstrates for the first time the presence of a functional tryptophan hydroxylase in a parasitic helminth and further suggests that the parasites are capable of synthesizing serotonin endogenously. A cDNA (Schistosoma mansonitryptophan hydroxylase; SmTPH) encoding a protein homologous to tryptophan hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of serotonin, was cloned from the human parasite Schistosoma mansoni. Bacterial expression of SmTPH as a histidine fusion protein produced soluble active enzyme, which was purified to apparent homogeneity and a final specific activity of 0.17 μmol/min/mg of protein. The purified enzyme was found to be a tetramer of approximately 240 kDa with a subunit size of 58 kDa. Several of the biochemical and kinetic properties of SmTPH were similar to those of mammalian tryptophan hydroxylase. Unlike the mammalian enzyme, however, SmTPH was found to be stable at 37 °C, itst½ being nearly 23 times higher than that of a similarly expressed rabbit tryptophan hydroxylase. A semiquantitative reverse transcription polymerase chain reaction showed that the level of SmTPH mRNA in a larval stage of the parasite (cercaria) is 2.5 times higher than in adult S. mansoni, suggesting possible differences in the level of enzyme expression between the two developmental stages. This study demonstrates for the first time the presence of a functional tryptophan hydroxylase in a parasitic helminth and further suggests that the parasites are capable of synthesizing serotonin endogenously. Tryptophan hydroxylase (TPH 1The abbreviations used are: TPH, tryptophan hydroxylase; SmTPH, S. mansoni TPH; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; TH, tyrosine hydroxylase; PAH, phenylalanine hydroxylase; bp, base pair(s); 5-HTP, 5-hydroxytryptophan; 5-HT, 5-hydroxytryptamine; BH4, (6R)-5,6,7,8-tetrahydrobiopterin; SL, spliced leader; tryptophan 5-monooxygenase; EC 1.14.16.4) catalyzes the hydroxylation ofl-tryptophan to 5-hydroxy-l-tryptophan (5-HTP). This reaction is the first and rate-limiting step in the biosynthesis of the monoamine neurotransmitter, serotonin (5-hydroxytryptamine; 5-HT) (reviewed in Ref. 1Mockus S.M. Vrana K.E. J. Mol. Neurosci. 1998; 10: 163-179Crossref PubMed Scopus (83) Google Scholar). TPH belongs to a family of aromatic amino acid hydroxylases that also includes the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH; EC 1.14.16.2) and phenylalanine hydroxylase (PAH; EC 1.14.6.1) (for reviews, see Refs. 2Hufton S.E. Jennings I.G. Cotton R.G. Biochem. J. 1995; 311: 353-366Crossref PubMed Scopus (184) Google Scholar, 3Kappock T.J. Cardonna J.P. Chem. Rev. 1996; 96: 2659-2756Crossref PubMed Scopus (290) Google Scholar, 4Kaufman S. Ribeiro P. Meyers R.A. The Encyclopedia of Molecular Biology. VCH Publisher, Inc., New York1996: 217-282Google Scholar). The three enzymes catalyze similar hydroxylation reactions and share a distinctive requirement for tetrahydrobiopterin (BH4) and non-heme ferrous iron as cofactors (2Hufton S.E. Jennings I.G. Cotton R.G. Biochem. J. 1995; 311: 353-366Crossref PubMed Scopus (184) Google Scholar). Studies of TPH have been hampered by the extreme instability of the enzyme (5Jequier E. Robinson D.S. Lovenberg W. Sjoerdsma A. Biochem. Pharmacol. 1969; 18: 1071-1081Crossref PubMed Scopus (172) Google Scholar, 6Friedman P.A. Kappelman A.H. Kaufman S. J. Biol. Chem. 1972; 247: 4165-4173Abstract Full Text PDF PubMed Google Scholar, 7Joh T.H. Shikimi T. Pickel V.M. Reis D.J. Proc. 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Neurosci. 1994; 5: 87-93Crossref PubMed Scopus (15) Google Scholar, 13Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar, 14Vrana K.E. Rucker P.J. Kumer S.C. Life Sci. 1994; 55: 1045-1052Crossref PubMed Scopus (30) Google Scholar, 15Yang X.J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar, 16D'Sa C.M. Arthur Jr., R.E. Kuhn D.M. J. Neurochem. 1996; 67: 917-926Crossref PubMed Scopus (41) Google Scholar, 17Banik U. Wang G.A. Wagner P.D. Kaufman S. J. Biol. Chem. 1997; 272: 26219-26225Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sequence analyses of TPH cDNAs derived from mammalian and other vertebrate species (13Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar,18Grenett H.E. Ledley F.D. Reed L.L. Woo S.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5530-5534Crossref PubMed Scopus (176) Google Scholar, 19Darmon M.C. Guibert B. Leviel V. Ehret M. Maitre M. Mallet J. J. Neurochem. 1988; 51: 312-316Crossref PubMed Scopus (120) Google Scholar, 20Boularand S. Darmon M.C. Ganem Y. Launay J.M. Mallet J. Nucleic Acids Res. 1990; 18: 4257Crossref PubMed Scopus (78) Google Scholar, 21Stoll J. Kozak C.A. Goldman D. Genomics. 1990; 7: 88-96Crossref PubMed Scopus (63) Google Scholar, 22Green C.B. Besharse J.C. J. Neurochem. 1994; 62: 2420-2428Crossref PubMed Scopus (68) Google Scholar, 23Florez J.C. Seidenman K.J. Barrett R.K. Sangoram A.M. Takahashi J.S. Brain Res. Mol. Brain Res. 1996; 42: 25-30Crossref PubMed Scopus (32) Google Scholar) revealed that TPH shares high overall sequence homology with the other two members of the hydroxylase family (1Mockus S.M. Vrana K.E. J. Mol. Neurosci. 1998; 10: 163-179Crossref PubMed Scopus (83) Google Scholar, 2Hufton S.E. Jennings I.G. Cotton R.G. Biochem. J. 1995; 311: 353-366Crossref PubMed Scopus (184) Google Scholar). More recently, deletion mutagenesis studies identified three main functional regions of TPH, including a conserved central core that comprises the catalytic domain (15Yang X.J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar, 16D'Sa C.M. Arthur Jr., R.E. Kuhn D.M. J. Neurochem. 1996; 67: 917-926Crossref PubMed Scopus (41) Google Scholar, 21Stoll J. Kozak C.A. Goldman D. Genomics. 1990; 7: 88-96Crossref PubMed Scopus (63) Google Scholar, 24Kumer S.C. Mockus S.M. Rucker P.J. Vrana K.E. J. Neurochem. 1997; 69: 1738-1745Crossref PubMed Scopus (34) Google Scholar, 25Mockus S.M. Kumer S.C. Vrana K.E. J. Mol. Neurosci. 1997; 9: 35-48Crossref PubMed Scopus (20) Google Scholar), a C-terminal intersubunit binding region responsible for the formation of enzyme tetramers (26Liu X. Vrana K.E. Neurochem. Int. 1991; 18: 27-31Crossref PubMed Scopus (30) Google Scholar, 27Mockus S.M. Kumer S.C. Vrana K.E. Biochim. Biophys. Acta. 1997; 1342: 132-140Crossref PubMed Scopus (25) Google Scholar), and a divergent N-terminal end. The latter is predicted to have a regulatory function (15Yang X.J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar, 24Kumer S.C. Mockus S.M. Rucker P.J. Vrana K.E. J. Neurochem. 1997; 69: 1738-1745Crossref PubMed Scopus (34) Google Scholar, 28Kuhn D.M. Arthur Jr., R. States J.C. J. Neurochem. 1997; 68: 2220-2223Crossref PubMed Scopus (46) Google Scholar) and may contribute to the instability of TPH (25Mockus S.M. Kumer S.C. Vrana K.E. J. Mol. Neurosci. 1997; 9: 35-48Crossref PubMed Scopus (20) Google Scholar) Serotonin, a well known neuroactive agent of the mammalian central nervous system and periphery (29Jacobs B.L. Azmitia E.C. Physiol. Rev. 1992; 72: 165-229Crossref PubMed Scopus (2139) Google Scholar), has been identified in every invertebrate phylum thus far investigated (30Weiger W.A. Biol. Rev. Camb. Philos. Soc. 1997; 72: 61-95Crossref PubMed Scopus (113) Google Scholar). In parasitic flatworms (platyhelminths), including the human bloodfluke, Schistosoma mansoni, 5-HT acts as an important regulator of motor activity and carbohydrate metabolism (for a review, see Ref. 31Davis R.E. Stretton A.O.W. Marr J.J. Muller M. Biochemistry and Molecular Biology of Parasites. Academic Press, Inc., San Diego1995: 257-287Crossref Google Scholar) and as such is critical for the survival of the parasite in the host. Immunofluorescence and histochemical studies have localized 5-HT in the central and peripheral nervous systems of the worm, as well as holdfast structures, body musculature, and reproductive structures (31Davis R.E. Stretton A.O.W. Marr J.J. Muller M. Biochemistry and Molecular Biology of Parasites. Academic Press, Inc., San Diego1995: 257-287Crossref Google Scholar). Earlier studies of 5-HT biosynthesis in parasitic helminths reported conflicting results. The failure to demonstrate TPH activity in crude tissue extracts of S. mansoni led some researchers to conclude that TPH is absent in this animal and that the parasite depends entirely on the host for a source of serotonin (32Bennett J.L. Bueding E. Mol. Pharmacol. 1973; 9: 311-319PubMed Google Scholar, 33Catto B.A. Ottesen E.A. Comp. Biochem. Physiol. C. 1979; 2: 235-242Crossref Scopus (36) Google Scholar, 34Cho C.H. Mettrick D.F. Parasitology. 1982; 84: 431-441Crossref PubMed Scopus (29) Google Scholar, 35Mansour T.E. Adv. Parasitol. 1984; 23: 1-36Crossref PubMed Scopus (99) Google Scholar, 36Wood P.J. Mansour T.E. Exp. Parasitol. 1986; 62: 114-119Crossref PubMed Scopus (13) Google Scholar). Although other authors have challenged these studies and presented preliminary evidence of 5-HT biosynthesis in related parasites (37Ribeiro P. Webb R.A. Mol. Biochem. Parasitol. 1983; 7: 53-62Crossref PubMed Scopus (28) Google Scholar, 38Ribeiro P. Webb R.A. Comp. Biochem. Physiol. C Comp. Pharmacol. 1984; 79: 159-164Crossref Scopus (33) Google Scholar, 39Chaudhuri J. Martin R.W. Donahue M.J. Int. J. Parasitol. 1988; 18: 341-346Crossref PubMed Scopus (7) Google Scholar), the question of whether TPH is present or absent in parasitic worms, in particular S. mansoni, remains largely unresolved. Here we report the cDNA cloning, purification, and functional characterization of tryptophan hydroxylase from S. mansoni(SmTPH). This study provides the first evidence that S. mansoni, and probably related parasites, possess the endogenous capability for de novo synthesis of 5-HT. When expressed inEscherichia coli, the purified SmTPH was highly active and, in contrast to the mammalian enzyme, very stable during purification and storage. SmTPH represents a potentially useful model for detailed biochemical studies of TPH structure and function. l-Tryptophan, 5-HTP, 5-HT,N-acetyl-5-HT, melatonin, p-chlorophenylalanine, isopropyl-β-thiogalactoside, dithiothreitol, dihydropteridine reductase, NADH, glycerol, and Sephacryl 200HR were purchased from Sigma. Tween 20, ferrous ammonium sulfate, and activated charcoal were from Fisher. [5-3H]-l-Tryptophan was from Amersham Pharmacia Biotech. (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and dopamine were from Research Biochemicals International. Aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and catalase were from Roche Molecular Biochemicals. All other chemicals were of the highest purity and quality from available commercial sources. A Puerto Rican strain of S. mansoniwas maintained as described previously (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar). Crude adult worm tissue extracts were prepared and used directly for TPH activity measurements as described earlier (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar). Total RNA was extracted from S. mansoni using the TRIzol reagent (Life Technologies, Inc.). Poly(A+) RNA from adult S. mansoni was purified from total RNA using oligo(dT)-cellulose columns (Amersham Pharmacia Biotech). A partial S. mansoni cDNA sequence (576 bp) homologous to other TPH sequences was isolated by homology RT-PCR. Oligonucleotide primers were synthesized based on a predicted genomic TPH sequence from the free-living nematode Caenorhabditis elegans(cosmid ZK1290; GenBankTM accession no. U21308) and used for PCR amplification of adult S. mansoni oligo(dT) reverse transcribed cDNA. The sense and antisense primers targeted a region of the predicted catalytic domain that is highly conserved among all aromatic amino acid hydroxylases (see Fig. 3). Primer sequences and RT-PCR conditions are described elsewhere (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar). The 5′-end of SmTPH was obtained using a RT-PCR method that targets the conserved 5′-end spliced leader (SL) sequence of S. mansonitranscripts (41Rajkovic A. Davis R.E. Simonsen J.N. Rottman F.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8879-8883Crossref PubMed Scopus (119) Google Scholar, 42Davis R.E. Hardwick C. Tavernier P. Hodgson S. Singh H. J. Biol. Chem. 1995; 270: 21813-21819Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, adult S. mansoni mRNA (0.5 μg) was reverse-transcribed with an oligo(dT) primer and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). One-tenth of the resulting cDNA was subjected to 30 cycles of PCR (30 s at 94 °C, 30 s at 53 °C, 90 s at 72 °C) in a 50-μl reaction containing 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 1.5 mm MgCl2, 0.2 mm dNTPs mix, 0.4 μm each primer, and 5 units of Taq DNA polymerase (Life Technologies, Inc.). Oligonucleotide SmSL (see Fig. 1), which corresponded to nucleotides 9–32 of the S. mansoni 36-nucleotide SL sequence (41Rajkovic A. Davis R.E. Simonsen J.N. Rottman F.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8879-8883Crossref PubMed Scopus (119) Google Scholar) was used as a sense primer, while oligonucleotide A (see Fig. 1) was used as an antisense primer. An aliquot (2 μl of 1:10 dilution) of the PCR product was similarly subjected to a second PCR reaction (25 cycles; same cycling parameters as above) using the same sense primer (SmSL) and a nested reverse primer (primer B; see Fig. 1). The resulting PCR product was gel-purified, cloned into the vector PCR 2.1 (Invitrogen), and sequenced by the dideoxy chain termination method. The 3′-end of SmTPH cDNA was amplified from an adult S. mansoni cDNA library in pcDNA3.1(+) (Invitrogen). An aliquot of the plasmid library (0.4 μg) was subjected to PCR (30 cycles) using a SmTPH-specific sense primer (primer C; see Fig. 1) and an antisense pcDNA3.1(+) vector-specific primer flanking the multiple cloning site (VSP1, 5′-GGAGGGGCAAACAACAGATGG-3′). The PCR cycling parameters were as above except for an annealing temperature of 55 °C. An aliquot of the PCR product was subjected to a second round of PCR amplification (25 cycles) using a nested SmTPH-specific sense primer (primer D; see Fig. 1) and pcDNA3.1(+) vector-specific-primer (VSP2, 5′-TAGAAGGCACAGTCGAGGC-3′). Amplified products were cloned into pCR2.1, and DNA was sequenced as before. For expression studies, the complete coding sequence of SmTPH was amplified by RT-PCR and subcloned into a T7 polymerase-based pET prokaryotic expression vector (43Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar). Oligo(dT) reverse transcribed cDNA was subjected to 35 cycles of PCR with primers that targeted the entire coding sequence of SmTPH (primers S and E; see Fig. 1) and a proofreading DNA polymerase (Pwo; Roche Molecular Biochemicals) according to the manufacturer's procedure. To facilitate further subcloning into the expression vector, enzyme restriction sitesNdeI and BamHI were incorporated at the 5′-end of the sense and antisense primers, respectively. The resulting PCR product was gel-purified, digested with NdeI and BamHI (Life Technologies, Inc.), and ligated into pET15b vector (Novagen), which was linearized by the same two restriction enzymes. Cloning into pET15b introduces an N-terminal oligohistidine fusion tag, which adds 20 amino acid residues of vector-derived sequence to the expressed SmTPH product. The final construct was confirmed by DNA sequencing of three independent clones and then used to transform E. coli host strain BL21(DE3)pLysS (Novagen). BL21(DE3)pLysS cells transformed with the SmTPH.pET15b construct were grown at 37 °C in LB-ampicillin-chloramphenicol medium to an A600 of ∼0.5–1.0 (log growth phase). Cultures were supplemented with 0.1 mm ferrous ammonium sulfate, as described previously (13Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar, 14Vrana K.E. Rucker P.J. Kumer S.C. Life Sci. 1994; 55: 1045-1052Crossref PubMed Scopus (30) Google Scholar, 15Yang X.J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar), and then induced with 1 mm isopropyl-β-thiogalactoside for 2.5 h at 30 °C. After induction, the cells were washed once with ice-cold 50 mm Tris-HCl (pH 8.0), pelleted by centrifugation, and stored frozen at −80 °C until used. For purification of recombinant SmTPH, cell pellets from 100 ml of induced bacterial cultures were thawed in 4 ml of 20 mm phosphate buffer (pH 7.4) containing 0.5m NaCl, 0.2% Tween 20, 5% glycerol, 10 mmimidazole, and a mixture of protease inhibitors (1 mmphenylmethylsulfonyl fluoride and 50 μg/ml each leupeptin and aprotinin). To promote cell lysis by the T7 resident lysogen, the cells were subjected to two cycles of rapid freeze-thawing followed by sonication on ice (seven pulses of 15 s separated by intervals of 30 s) using a vibra cell sonicator (Sonics and Material, Danbury, CT) set at 20% maximal power. Cell lysates were centrifuged at 12,000 × g for 15 min and 4 °C. The resulting pellet was resuspended in 2.5 ml of the same buffer and similarly sonicated and centrifuged as above. The two supernatants were pooled and used for direct enzymatic assays and for subsequent purification of the expressed enzyme. Recombinant SmTPH was purified by immobilized metal (nickel) affinity chromatography (44Linder P. Guth B. Wulfing C. Krebber C. Steipe B. Muller F. Pluckthun A. Methods Companion Methods Enzymol. 1992; 4: 41-56Crossref Scopus (70) Google Scholar) using the HisTrap kit (Amersham Pharmacia Biotech) for purification of histidine-tagged proteins, as described previously (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar). Excess imidazole was removed by gel filtration through a Sephadex G-25 column (PD-10; Amersham Pharmacia Biotech), and the purified enzyme was stored in 50 mm HEPES (pH 7.5) containing 0.2m NaCl, 10% glycerol, 0.05% Tween 20, and 1 mm dithiothreitol. Purified enzyme preparations (0.2 mg/ml) were stable for at least 4 days at 4 °C and could be stored at −80 °C for at least 1 month with no significant loss in activity. TPH activity was measured using the tritiated water release method (45Beevers S.J. Knowles R.G. Pogson C.I. J. Neurochem. 1983; 40: 894-897Crossref PubMed Scopus (23) Google Scholar, 46Vrana S.L. Dworkin S.I. Vrana K.E. J. Neurosci. Methods. 1993; 48: 123-129Crossref PubMed Scopus (39) Google Scholar) with few modifications. The standard assay was performed in a 100-μl reaction of 50 mm HEPES (pH 7.5) containing 400,000 cpm ofl-[5-3H]Tryptophan and enough unlabeledl-tryptophan to make a final concentration of 100 μm, 0.2 mg/ml catalase, 0.4 mm NADH, 10 milliunits of dihydropteridine reductase, 10 μmdithiothreitol, and either purified SmTPH (0.6 μg) or crude adultS. mansoni tissue extract (50–100 μg of protein). The reaction was started with the addition of 200 μmBH4, unless indicated otherwise, and the samples were incubated for 10 min at 37 °C. Preliminary experiments revealed that TPH activity increased linearly up to 12 min of incubation under these conditions. The reaction was terminated by the addition of 1 ml of activated charcoal (7.5% (w/v) in 1 m HCl) to each sample. After centrifugation (2000 × g for 10 min), aliquots of the supernatants were radioassayed in 10 ml of scintillation mixture (ICN). Enzyme activity data were analyzed using Lineweaver-Burk plots or by computer-assisted, nonlinear curve fitting to the Michaelis-Menten model. All kinetic parameters (Kmand apparent Km (S0.5)) were determined using the program Enzyme Kinetics (version 1.C; DogStar Software) and were obtained from two to three independent experiments, each performed in duplicates or triplicates. The stability of SmTPH was assessed in comparison with that of recombinant rabbit brain TPH similarly expressed in E. coli. In preparation for these experiments, the complete coding sequence of rabbit brain TPH cDNA (13Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar) was subcloned into pET15b and expressed as a histidine-tagged protein in BL21(DE3)pLysS E. coli. Bacterial cells expressing rabbit TPH or SmTPH were lysed, as described above, and the corresponding soluble fractions containing expressed enzyme were passed through a Sephadex G25 PD10 (Amersham Pharmacia Biotech) column equilibrated with the same HEPES buffer described earlier. Stability was measured as a function of time according to the procedure of Mockus et al.(25Mockus S.M. Kumer S.C. Vrana K.E. J. Mol. Neurosci. 1997; 9: 35-48Crossref PubMed Scopus (20) Google Scholar). Briefly, aliquots (100 μg of protein) of the crude SmTPH or rabbit TPH extracts were preincubated at 37 °C for varying lengths of time (0, 10, 20, 40, and 80 min) and then assayed for TPH activity. The data were calculated as a percentage of the initial level of activity (t = 0) for each enzyme. Semiquantitative RT-PCR was employed for the determination of SmTPH mRNA levels. Total RNA (∼2 μg) from two developmental stages of S. mansoni (cercaria and adults) were subjected to DNase I treatment (amplification grade; Life Technologies, Inc.) followed by a standard RT-PCR reaction (1 min at 94 °C, 14–36 cycles of 30 s at 94 °C, 30 s at 53.5 °C, 60 s at 72 °C, and 7 min at 72 °C) using primers (sense, primer D; antisense, primer F; see Fig. 1) that amplify an 873-bp cDNA product from SmTPH. PCR was standardized by simultaneous amplification of a constitutively expressed control housekeeping gene, S. mansoni α-tubulin (47Webster P.J. Seta K.A. Chung S.C. Mansour T.E. Mol. Biochem. Parasitol. 1992; 51: 169-170Crossref PubMed Scopus (20) Google Scholar, 48Mei H. LoVerde P.T. Exp. Parasitol. 1997; 86: 69-78Crossref PubMed Scopus (107) Google Scholar), as described previously (49Kinoshita T. Imamura J. Nagai H. Shimotohno K. Anal. Biochem. 1992; 206: 231-235Crossref PubMed Scopus (194) Google Scholar). The PCR primers used for the amplification of the 558-bp S. mansoni α-tubulin cDNA fragment were as follows: sense, 5′-CTTATCGTCAACTTTTCCATCC-3′; antisense, 5′-GGAAGTGGATACGAGGATAAGG-3′ (modified from Ref. 48Mei H. LoVerde P.T. Exp. Parasitol. 1997; 86: 69-78Crossref PubMed Scopus (107) Google Scholar). Standard curves were generated to ensure that the PCR assay was in the exponential phase of synthesis after 34 cycles for SmTPH or 24 cycles for α-tubulin. The resulting PCR products were cloned in PCR2.1 and confirmed by DNA sequencing. Densitometric analysis of the ethidium bromide-stained RT-PCR products were performed with the NIH Image program version 1.61 (Bethesda, MA). Size exclusion chromatography of purified SmTPH was performed on a Sephacryl-200HR gel filtration column (10-mm inner diameter × 50 cm; Bio-Rad), as described previously (27Mockus S.M. Kumer S.C. Vrana K.E. Biochim. Biophys. Acta. 1997; 1342: 132-140Crossref PubMed Scopus (25) Google Scholar). Protein concentrations were measured by the method of Bradford (50Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar), using the Bio-Rad protein assay kit and bovine serum albumin as a standard. Reducing SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (51Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) using precast 10% acrylamide gels from Novex, Inc. For Western blot analysis of SmTPH, aliquots of purified enzyme (0.5–1 μg) were electrophoresed as above, transferred onto nitrocellulose (52Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and reacted with a sheep polyclonal antibody (1:500 dilution) raised against rabbit TPH (Chemicon International) followed by a peroxidase-conjugated rabbit anti-sheep IgG (Pierce) as the secondary antibody (1:1000 dilution). A partial TPH sequence was first obtained by RT-PCR using oligo(dT) reverse-transcribed S. mansoni cDNA and C. elegans primers (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar) that targeted a region conserved among all aromatic amino acid hydroxylases. A 576-bp product was sequenced and found to have high homology with TPH sequences from other species. The missing 5′- and 3′-ends were subsequently obtained by an anchored PCR-based strategy. The 5′-end was amplified in a RT-PCR reaction that targeted the conserved SL sequence of S. mansoni (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar, 41Rajkovic A. Davis R.E. Simonsen J.N. Rottman F.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8879-8883Crossref PubMed Scopus (119) Google Scholar). A 741-bp product corresponding to the 5′-end of TPH was sequenced and found to carry a complete S. mansoniSL (nucleotides 9–36 of the SL sequence; see Fig. 1) including the last four nucleotides, which were not part of the SmSL primer used in the anchored PCR reaction. This finding suggests that SmTPH is trans-spliced at the 5′-end to the S. mansoni SL, just as described previously for several other S. mansoni cDNAs (40Hamdan F.F. Ribeiro P. J. Neurochem. 1998; 71: 1369-1380Crossref PubMed Scopus (22) Google Scholar, 41Rajkovic A. Davis R.E. Simonsen J.N. Rottman F.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8879-8883Crossref PubMed Scopus (119) Google Scholar, 42Davis R.E. Hardwick C. Tavernier P. Hodgson S. Singh H. J. Biol. Chem. 1995; 270: 21813-21819Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The 3′-end of SmTPH was PCR-amplified directly from a S. mansoni cDNA plasmid library, using TPH-specific and vector-derived primers (see Fig. 1). The resulting product (1000 bp) contains a potential polyadenylation sequence (AATATA) (53Birnstiel M.L. Busslinger M. Strub K. Cell. 1985; 41: 349-359Abstract Full Text PDF PubMed Scopus (946) Google Scholar) upstream of a poly(A) tail and thus is presumed to represent the 3′-end of the full-length transcript. Fig. 1 shows the nucleotide and predicted amino acid sequence of SmTPH. The composite cDNA reveals a single open reading frame of 1494 bp encoding a predicted protein of 497 amino acids with a calculated molecular mass of 58 kDa. Protein sequence analysis revealed the presence of two consensus sites (Ser151 and Thr130) for phosphorylation by the Ca2+/calmodulin-dependent protein kinase type II and a consensus leucine zipper motif (SmTPH amino acid positions 358–377). BLAST analysis (54Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71458) Google Scholar) of the predicted protein sequence indicated that SmTPH is highly homologous to tryptophan hydroxylase from other species. The dendrogram in Fig. 2 shows that SmTPH is more related to TPH sequences than to those of the other two aromatic amino acid hydroxylases, TH and PAH. Based on pairwise CLUSTAL protein alignments (55Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar), SmTPH shares high amino acid sequence homology (65–67%) with vertebrate TPH sequences (13Tipper J.P. Citron B.A. Ribeiro P. Kauf" @default.
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• W2091879423 title "Characterization of a Stable Form of Tryptophan Hydroxylase from the Human Parasite Schistosoma mansoni" @default.
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