The identification and characterization of two unique membrane-associated molecules of African trypanosomes




Stebeck, Caroline Elizabeth

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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 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.



African trypanosomiasis