The primary structure of a 38 kDa protein isolated from membrane preparations of
African trypanosomes (Trypanosoma brucei rhodesiense) was determined by protein and
DNA sequencing. Searching of the protein database with the trypanosome translated
amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from
various prokaryotic and eukaryotic organisms as the optimal scoring protein.
Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence
identity with the corresponding enzyme from the prokaryote Escherichia coli. Using
recombinant DNA techniques, the trypanosome molecule was expressed in Escherichia
coli and found to be enzymatically active, thus confirming the identity of the molecule as
an NAD+-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody
specific for the 38 kDa protein was used to localize the enzyme to glycosomes. The
enzyme has a pi of 9.0, a net charge of +9 at physiological pH and contains the
peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of
glycosomal enzymes. Amino acids predicted to be involved in the NAD+-dependent
glycerol 3-phosphate dehydrogenase active site have diverged from those of the
mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit
muscle showed that the Km values of the two enzymes are different The data suggests
that the trypanosome protein may be a candidate target for rational drug design. Northern
and Southern blot analyses showed that the trypanosome NAD+-dependent glycerol 3-
phosphate dehydrogenase was translated from a single transcript and that only two gene
copies exist thus making this molecule an attractive target for knockout mutagenesis.
A second molecule, an abundant 11 kDa membrane protein, was also purified from
African trypanosomes. This protein cross-reacted with monoclonal antibodies originally
generated against the lipophosphoglycan-associated protein of Leishmania donovani.
Immunoblot analysis showed that the 11 kDa molecule was present in a variety of species
of kinetoplastids. It was found in several species and subspecies of African
trypanosomes and was present in low amounts in bloodstream forms and in larger
amounts in procyclic, epimastigote and metacyclic life cycle stages. The molecule was
present in procyclic trypanosome membranes at approximately [special characters omitted] molecules
per cell. Its wide distribution in kinetoplastids and its membrane disposition suggested a
name for this class of molecules (kinetoplastid membrane protein-11) and for the molecule
characterized in this thesis (trypanosome kinetoplastid membrane protein-11).
The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma
brucei rhodesiense by organic solvent extraction and octyl-Sepharose chromatography
and a 14 amino acid internal peptide sequence was obtained by gas phase
microsequencing. This sequence matched a translated Leishmania donovani kinetoplastid
membrane protein-11 sequence, thus suggesting the use of the Leishmania sequence as a
probe to select for the Trypanosoma gene. Screening of a trypanosome cosmid library
with the Leishmania probe, in combination with a series of polymerase chain reaction
amplifications from both genomic DNA and cDNA, allowed the determination of the
entire DNA sequence and corresponding translated amino acid sequence of the
trypanosome kinetoplastid membrane protein-11. The 92 amino acid sequence showed
18 percent sequence divergence from the corresponding molecule of the related
kinetoplastid Leishmania donovani donovani^ including one key amino acid at position 45
which may be of functional relevance. The secondary structure of the trypanosome
molecule was predicted to form two amphipathic helices connected by a random-coil
segment, and suggests that it would interact with lipid bilayers in the parasite cell
membrane. Northern and Southern blot analyses using the T.b. rhodesiense ViTat 1.1
clone showed that the trypanosome molecule was translated from a single transcript and
that there was only a single gene copy, thus making this molecule an attractive target for
knockout mutagenesis. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9742 |
| Date | 19 July 2018 |
| Creators | Stebeck, Caroline Elizabeth |
| Contributors | Pearson, Terry W. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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