McKie, Amanda Elaine
Molecular and biochemical characterisation of Methionine γ-lyase from Trichomonas vaginalis.
PhD thesis, University of Glasgow.
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Two methionine -lyase gene homologues, mgl1 and mgl2 have been isolated from Trichomonas vaginalis using a degenerate oligonucleotide/PCR approach. Degenerate oligonucleotides designed against cystathionine -lyase from yeast, rat and human were used in the PCR experiments. mgl1 and mgl2 are present at single copy in the T. vaginalis genome and are expressed to give 1.3kb mRNAs. The two genes have extremely short 5' untranslated regions. The predicted molecular mass of MGL1 and MGL2 are 42.9 and 43.1 kDa, respectively. High homology exists at the amino acid level between the two T. vaginalis methionine -lyase gene homologues and methionine -lyase from Pseudomonas putida and cystathionine -lyase from a range of organisms and other related sulphur amino acid-metabolising enzymes.
The two methionine -lyase homologues were cloned into expression vectors and recombinant proteins purified and subsequently characterised. Biochemical characterisation of rMGL1 and rMGL2 revealed that both recombinant proteins were able to break down methionine, catabolise homocysteine at high rate and also able to metabolise cysteine and O-acetyl L-serine. Interestingly, the recombinant proteins were not able to break down cystathionine. The two proteins were expressed in T. vaginalis, as judged by ~43 kDa proteins being detected in T. vaginalis lysates and soluble extracts by Western blot analysis.
Tritrichomonas foetus and Tritrichomonas augusta do not possess the ability to breakdown homocysteine and are thought not to contain methionine -lyase. Interestingly, however, antibodies raised against rMGL1 and rMGL2 recognised proteins of various molecular weights and upon immunostaining with intact parasites, it is probable that the Golgi apparatus of these parasites were recognised by the anti rMGL1 and rMGL2 sera. For T. vaginalis, immunostaining using the anti rMGL1 and rMGL2 sera revealed a staining of the nucleus and a more general staining of the cytoplasm of these parasites.
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