Computational protocol: Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

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Protocol publication

[…] Using the Saccharomyces cerevisiae EK gene sequence as a query, two putative C/EK (choline/ethanolamine kinase) genes were identified in the T. brucei genome database (http://www.genedb.org) using tBlastN. The two putative ORFs (Tb11.18.0017 for TbC/EK1, and Tb927.5.1140 for TbC/EK2) together with ∼350 bp of their 5′- and 3′-UTRs (untranslated regions), were amplified from T. brucei strain 427 genomic DNA using Pfu DNA polymerase and the forward and reverse primers 5′-ATAAGTAAGCGGCCGCCCGCCTAAGTTAGAAGTTGCGCT-3′ and 5′-ATAAGTAAGCGGCCGCTCCAATAGCTCCAGGGAAGGAAAGGGACG-3′ for TbC/EK1; and 5′-ATAAGTAAGCGGCCGCAAGTGCGTTGTGCAGGTCGGCGACGGT-3′ and 5′-ATAAGTAAGCGGCCGCCGTTGGAAGGAGGAAAACGGCCGAGG-3′ for TbC/EK2. The resulting 2.1 kb (TbC/EK1 and UTRs) and 3.7 kb (TbC/EK2 and UTRs) fragments were cloned into pCR®-Blunt-II TOPO®. Clones were sequenced and compared with the annotated GenBank® sequences. A BamHI restriction site, internal to the TbC/EK1 ORF was silenced by site-directed mutagenesis using the forward primer 5′-GTGTTGAGGGGGATAAGCGAATCCATCGCATGGTTCAGC-3′, the reverse primer 5′-GCTGAACCATGCGATGGATTCGCTTATCCCCCTCAACAC-3′ and the QuikChange® site-directed mutagenesis kit (Stratagene).The TbC/EK1 ORF was PCR-amplified from the TOPO® construct with Pfu polymerase using the forward and reverse primers 5′-GGAATTCCATATGATGGAGGTGGCTGTGGGGCAC-3′ and 5′-CGCGGATCCGCGTTATGAAGATGCACTAAACTC-3′ respectively. The amplicon was purified (QIAquick PCR purification kit; Qiagen), ligated into pCR®-Blunt II TOPO® and sequenced. Using the NdeI and BamHI restriction sites (underlined in primer sequences), the putative TbC/EK1 was ligated into the expression vector pET-15b (Novagen) modified with a TEV (tobacco etch virus) protease cleavage site (in place of a thrombin cleavage site) using the same restriction sites generating the pET-15bTEV-TbC/EK1 construct. The same procedure was employed to ligate the putative TbC/EK2 into the same vector using the forward and reverse primers 5′-GGAATTCCATATGATGGCATTACGACCGTTCCCG-3′ and 5′-CGCGGATCCGCGTCAGGAAAGAAGCCCCTTCTC-3′ respectively.The D287A and D267A mutants of TbC/EK1 were generated using the QuikChange® site-directed mutagenesis kit with the forward and reverse primers 5′-GGCGCACTGAAGATTATTGcCTTCGAGTATGCAAAACG-3′ and 5′-CGTTTTGCATACTCGAAGgCAATAATCTTCAGTGCGCC-3′ respectively for the D287A mutant and the forward and reverse primers 5′-GAGTACGTGCCACAATGcCCTGCTCAGCGGC-3′ and 5′-GCCGCTGAGCAGGgCATTGTGGCACGTACTC-3′ respectively for the D267A mutant (the lower case letters in the primers highlight the mutated bases). All DNA sequencing was performed by The Sequencing Service (College of Life Sciences, University of Dundee; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye version 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. [...] EK or CK activity was measured by a spectrophotometric coupled assay as described previously []. The assay contained 50 mM Mops (pH 7.8), 150 mM KCl, 6 mM MgCl2, 0.5 mg/ml BSA, 1 mM phosphoenolpyruvate, 0.5 mM NADH, 7 units/ml pyruvate kinase, 32 units/ml lactate dehydrogenase and 2 μg/ml TbC/EK1 or 1 μg/ml TbC/EK2. Concentrations of ATP and ethanolamine (or choline) were varied, and the rate of the reaction was monitored by the decrease in absorbance at 340 nm (a consequence of NADH oxidation) in a Shimadzu UV-1601 spectrophotometer. The Enzyme Kinetics module of SigmaPlot (SPSS) was used to analyse and fit the kinetic data.The pH optimum was assessed by measuring TbC/EK1 and TbC/EK2 activities at various pHs: sodium acetate (pH 5.0), Bis-Tris/propane or Mops (pH 6.5), Tris/HCl (pH 7.0, 8.0 and 8.5), Mops (pH 6.0, 7.0, 7.4 and 7.8) and glycine (pH 9.0). Various ethanolamine and choline analogues were tested as substrates at least in triplicate on 96-well plates using a SpectraMAX 340PC plate reader (Molecular Devices), and those deemed as non-substrates were tested as inhibitors by pre-incubation at 1 mM for 5 min before the addition of ethanolamine or choline (0.1 mM).Direct EK and CK activity assays were performed by assessing the production of PtdEtn or PtdCho, using a modified method of Kim et al. []. Briefly, 2 μg of purified protein was incubated with a reaction mixture (total volume of 50 μl) of 100 mM Mops (pH 7.8), 6 mM MgCl2 and 5 mM ATP, with either 2 mM ethanolamine and 0.2 μCi of [3H]ethanolamine or 2 mM choline and 0.2 μCi of [3H]choline at 37 °C for 20 min and quenched by boiling at 100 °C for 5 min. Substrates and products were separated by HPTLC (high-performance TLC) using silica 60 plates with methanol/0.6% NaCl/ammonium hydroxide (10:10:1, by vol.) as solvent. Radiolabelled species were detected by fluorography at −70 °C after spraying with En3Hance and using Kodak XAR-5 film with an intensifying screen. Unlabelled ethanolamine and PtdEtn standards were run in parallel and visualized by spraying with ninhydrin (0.2% in water-saturated butan-1-ol). Unlabelled PtdCho was run in parallel and visualized by iodine staining. […]

Pipeline specifications

Software tools TBLASTN, SigmaPlot, SPSS
Databases GeneDB
Applications Miscellaneous, Amino acid sequence alignment
Organisms Trypanosoma brucei
Chemicals Adenosine Triphosphate, Carbon, Choline, Phosphatidylcholines, Phosphorylcholine, Glycosylphosphatidylinositols, Ethanolamine