Parasite strains and Reagents
Toxoplasma gondii strains used; virulent type I RH-YFP strain (48) and avirulent, cyst forming type III NED (a gift from Johan Lindh). All chemicals were purchased from Sigma, with the exception of 6-MPH4 from Schircks laboratories (Jona, Switzerland), Talon resin from Clontech and anti-tyrosine hydroxylase monoclonal antibody from Calbiochem (San Diego, CA).
Toxoplasma gondii parasites were maintained in continuous passage in Hs27 human foreskin fibroblast (HFF) cell monolayers (ECACC number 94041901). Parasites were grown in DMEM supplemented with 10% foetal bovine serum (Invitrogen, Paisley, UK) and passaged between 4 and 15 times prior to bradyzoite induction. Bradyzoites were induced in culture using the high (pH 8.1) pH shock method (49). The number of parasitized cells, vacuoles and the number of parasites per vacuole were scored every 24 hours to monitor differentiation. Four parasitized monolayers were harvested every 24 hours by trypsinization and parasites purified by passage through a 21 gauge needle and washing in phosphate-buffered saline (PBS). Parasites were enumerated and pellets were resuspended in Qiagen RNeasy kit RTL buffer prior to RNA extraction. For Western blotting, parasites were harvested as in Roberts et al. (50). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-rat-tyrosine hydroxylase monoclonal antibody.
Prediction of the T. gondii tyrosine hydroxylase genes
The SHARKhunt search programme, part of the metaSHARK package for automated reconstruction of metabolic pathways, was used to predict enzymes encoded in the T. gondii
genome (sequence freely provided to the community by the Welcome Trust Sanger Institute, Cambridge, UK and the Institute for Genomic Research (TIGR), USA) (15–17). SHARKhunt uses HMMER profile hidden Markov models (HMM's) based on the PRIAM library of polypeptide profiles (51) to search a set of DNA sequences (finished chromosomes, contigs or expressed sequence tags) for potential enzyme encoding genes (52). The current enzyme collection contains 2562 profiles, covering 1967 enzymatic functions as defined by E.C. number. Regions of DNA showing some similarities to these profiles are analysed in detail using GeneWise (53), which reconstructs a putative gene structure wherever possible. Finally a confidence score in the form of an E-value is calculated to represent the similarity between each predicted protein product and its corresponding profile model. Applying this software resulted in a set of genes whose sequences were compared with sequences of predicted signal peptide containing proteins (based on SignalP) in the T. gondii
genome resource ToxoDB providing a list of potentially secreted proteins (www.toxodb.org
, (18,19)). SHARKhunt predicted a tyrosine hydroxylase in a reconstruction of the pathway for de novo
dopamine biosynthesis from the T. gondii
genome that was subsequently found to be encoded by two genes via ToxoDB. These genes are mapped to different locations on chromosome V in the T. gondii
genome at ToxoDB. The genomes of the apicomplexan parasites Neospora caninum
(Wellcome Trust Sanger Centre and the University of Liverpool), Plasmodium falciparum
(54), Plasmodium vivax
(The Institute for Genomic Research), Theileria annulata
(55), Babesia bovis
(56), Cryptosporidium parvum
(57) and Eimeria tenella
(Wellcome Trust Sanger Centre and BBSRC Institute for Animal Health) were also analysed for presence of an aromatic amino acid hydroxylase.
Sequence alignments were created using Muscle multiple alignment software (58). The signal peptide is predicted by SignalP (59) to reside between positions 1 and 24 based on eukaryotic networks prediction and HMM of eukaryotic models prediction (60).
Total RNA was extracted from purified T. gondii cell pellets using Qiagen RNeasy kit according to the manufacturers' instructions, and cDNA synthesis performed using Superscript II reverse transcriptase (Invitrogen). Quantity and quality of the RNA was monitored by spectrophotometry and by gel electrophoresis. To confirm stage conversion to bradyzoites, the RNA expression of three marker genes was also followed throughout the experiment. These were SAG 1 (primer sequences 5′-CGACAGCCGCGGTCATTCTC-3′ and 5′-CGACAGCCGCGGTCATTCTC-3′) (28), SAG 4, (primer sequences 5′-TGGACCTACGATTTCAAGAAGGC-3′ and 5′-GCTGCGAGCTCGACGGGCTCATC-3′) (29) and BAG 1 (primer sequences 5′-TCCGCCGGGAGCTTGTCCACC-3′ and 5′-GCAAGTCAGCCAAAATAATCA-3′) (30). Primers were designed to detect two housekeeping genes, actin (primer sequences 5′-CGAGCTGGTCAGTTCCTCAT-3′ and 5′-CATCGGGCAATTCATAGGAC-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (primer sequences 5′-GTATTGGCCGTCTGGTGTTC-3′ and 5′-CGTGGACCGAGTCGTATCTC-3′) for standardization. Quantitative PCR was carried out using Abgene SYBR green master mix (Rockford, IL) in a Bio-Rad I-cycler (Hercules, CA) for 40 cycles. Melt curve analysis was used to confirm the absence of primer-dimer formation or genomic DNA (Bio-Rad I-cycler QI software version 3.0). All expression profiles were standardised against parasite GAPDH and parasite Actin as housekeeping genes. All primers were specific to parasite cDNA and did not amplify HFF cDNA.
PCR amplification and open reading frame confirmation of TgAaaH 1 & 2
cDNA from RH and NED parasites was synthesized as above. For both genes 3′ RACE was used to confirm the 3′ UTR terminus, and walking up the gene with primers confirmed the 5′ transcription initiation sites (). The second MET codon within the ORF (open reading frame) of TgAaaH1 is assumed to be the translation start site due to conservation with TgAaaH2 and a conserved predicted signal peptide. This is also found in the predicted coding region of the N. caninum gene. PCR amplification using primers within the 5′ and 3′UTR of both genes allowed specific amplification of each transcript. Both genes were subcloned into pGEM-T Easy vector (Promega, Madison, WI) for sequencing. There were no coding differences between the RH and NED genes.
Expression and purification of recombinant proteins
Restriction sites for Pst I and Not 1 were added to the termini of both full length ORFs by PCR using Phusion polymerase (Invitrogen) from the pGEM-T constructs. Primers were: forward primer 5′-CTGCAGATGTGGCAGGCCATTTTCGCTG-3′, reverse primer (TgAaaH1) 5′-GCGGCCGCTAGAACCTGAGGGAAACGGG-3′ and reverse primer (TgAaaH2) 5′-GCGGCCGCTAGATCTTGAGGGAGACGGG-3′. The PCR products were again sub-cloned into pGEM-T easy vector (Promega), digested with Pst I and Not I restriction enzymes and inserted into pET 45b expression vector (Novagen, Madison, WI) with Promega ligase. Vectors containing the correct insert were confirmed by sequencing and double digest with Pst I/Not I and with Bam HI only. These constructs were named Apo-TgAaaH1 and Apo-TgAaaH2.
Deletion constructs were generated by PCR with Phusion polymerase using primers beginning at residue 23 (5′- GGTTCTGCAGGCTGTCCCCCAAGAATC-3′) to residue 239 (5′-CTGCAGGTCCCGTGGTTCCCTCGGTCT-3′) from both TgAaaH 1 and TgAaaH2 cloned in pGEM-T plasmids, generating a terminal Pst1 site and an in-frame start codon. The PCR products were cloned into pGEM-T easy before sub-cloning into pET 45b as above. Deletion of the first 23 residues removed the region predicted to be a signal peptide (), these were then referred to as TgAaaH 1&2. Deletion of the first 239 residues results in constructs beginning at the VPWFPR motif at the start of the putative catalytic domain (20, 61, 62), these were then named Δ239TgAaaH 1 & 2. All constructs were sequenced in pET 45b prior to expression ().
All pET 45b constructs were transformed by electroporation into BL21 GOLD (DE3) pLysS E. coli (Novagen). Cultures were grown for ~4 hrs at 37°C in TB media supplemented with 10 µM ferrous ammonium sulphate to an OD600 of ~0.8, induced with 0.1 mM IPTG and shaken for 20 hrs at 18°C (TgAaaH) or 28°C (Δ239TgAaaH). Cultures were harvested by centrifugation at 10,000×g for 5 min and pellets stored at −70°C overnight. All cell pellets were resuspended in 50 mM Tris-HCL pH 7.5, plus 0.2% Triton-X 100 and EDTA-free protease inhibitors (Roche, Nutley, NJ). These were sonicated for 30 sec with 30 sec rest three times on ice. Extracts were spun at 20,000×g for 20 min at 4°C and the soluble fraction removed. This was repeated two times with ten cycles of 30 s sonication/30 s rest on ice. The resulting soluble fraction was passed over a Talon affinity resin column (Clontech) and eluted with 500 mM imidazole. The imidazole was removed by dialysis for 2 hrs at 4°C against 50 mM Tris-HCL pH 7.5, plus 0.2% Triton-X100. Coomasie-stained SDS-PAGE electrophoresis and Western blotting with anti-rat-tyrosine hydroxylase monoclonal antibody and anti-His-tag antibody assessed the purity of each protein. As tyrosine hydroxylases contain an iron atom embedded in the structure that is required for catalytic activity, we increased the Fe2+ concentration of the expression media by the addition of ferrous ammonium sulphate to ensure that all the recombinant enzyme would contain Fe2+ (63–65). The additional iron increased the percentage of soluble protein suggesting that the presence of the central iron atom may be required for correct folding in this expression system. The addition of 0.2% Triton-X 100 and low salt concentrations also enhanced the solubility as seen with previous recombinant tyrosine hydroxylase enzymes (66).
Constructs for the expression of rat tyrosine hydroxylase (rat-WT-TyrH) and the catalytic domain of rat phenylalanine hydroxylase (rat-Δ117PheH) in pET 23d, were a kind gift from Paul Fitzpatrick. Both proteins were expressed without a tag and enriched by step-wise ammonium sulphate precipitation (61).
Assays were developed and standardized using recombinant proteins from expression of rat-WT-TyrH and rat-Δ117PheH. Protein concentration was determined using the Bradford assay (67). All assays were initiated by the addition of 10 µg enzyme. As the reproducibility of biopterin-dependent enzyme kinetics are greatly affected by the instability of the biopterin cofactor, 6-MPH4 was used and checked prior to assay by assessing the 240–300 nm spectrum. Assays were 10 min as biopterins are unstable in air and prolonged incubation with BH4 ((6R)-tetrahydropterin) will inactivate these enzymes (68, 69). The hydroxylation of tyrosine was measured by the method of Fitzpatrick (25) with the modifications of Royo et al. (26). Conditions were 25°C, pH 7.5, 10 mM HEPES, 1 mM DTT, 100 µg/ml catalase and 10 µM ferrous ammonium sulphate. Assays were terminated at 10 min by the addition of 15 µl 50% HCL, 200 µl 12.5% sodium nitrite and 200 µl 12.5%. NaOH (70 µl 3 M) was then added to each reaction and the absorbance at 500 nm read precisely 10 sec later. All assays were initiated by the addition of the enzyme. Additionally, the hydroxylation of phenylalanine was assayed by direct measurement of the production of tyrosine at 275 nm according to the method of Pember et al. (70) with the modifications of Daubner et al. (71). Conditions were 25°C, pH 7.5, 80 mM HEPES, 5 mM DTT, 60 µg/ml catalase and 10 µM ferrous ammonium sulphate. Tyrosine and dopa production was also detected by reverse phase HPLC of tritiated products. L-[2,3,4,5,6-3H]phenylalanine (25 µCi; final chemical concentration 1 µM) or 25 µCi L-[2,3,5,6-3H]tyrosine (final chemical concentration 1.25 µM) (GE Healthcare) was added to 10 µg recombinant protein in 200 µl reaction volumes. Reaction conditions were 500 µM 6-MPH4, 50 mM HEPES, 20% glycerol, 0.25% Triton-X 100 and EDTA free protease inhibitors (Roche) at pH 7.5. Reactions were initiated by the addition of enzyme and allowed to proceed for 4 hrs at 37°C. Reactions were stopped by freezing in liquid nitrogen and stored at −80°C prior to HPLC analysis.
In preparation for HPLC 50 µl of each assay mix was added to 210 µl 0.1% TFA (trifluroacetic acid). To this was added 5 µl of 1 mg/ml dopa, tyrosine and phenylalanine as standards. Samples were spun at 13000×g in a bench top microfuge to pellet the precipitated protein. Products were separated by reverse phase HPLC on a Synergi 4u Hydro 80A column (150×4.60 mm, Phenomenex, Macclesfield, UK) with an elution gradient of 0–60% acetonitrile with 0.1% TFA over 20 min at a flow rate of 1 ml/min. Fractions (0.5 ml) were collected every 30 sec from 6 min-18 min for each sample. Each fraction was counted for 1 min in 3 ml liquid scintillant (Packcard, Minnesota, USA) on a Wallac 1450 Microbeta Trilux counter using default settings.
For kinetic calculations an assay measuring the oxidation of 6-MPH4 using a coupled reaction measuring the decrease in absorbance at 340 nm due to NADH oxidation (61) was used as this assay has been validated for use in several previous studies (20,25,66). Conditions were 32°C, pH 7.5, 10 µg purified protein, 50 mM HEPES, 100 µg/ml catalase, 500 µM NADH, 10 µM ferrous ammonium sulphate, 0.2 u/ml sheep liver dihydropteridine reductase. For KmTyr/KmPhe measurements concentrations of amino acids were varied from 0–200 µM with 500 µM 6-MPH4. For Km6-MPH4 concentrations of 6-MPH4 were varied from 0–500 µM with 50 µM of each amino acid separately.
Kinetic data was fitted to the Michaelis-Menten equation using Graphpad PRISM (San Diego, California).