The phosphoglycerate kinases from Trypanosoma brucei. A comparison of the glycosomal and the cytosolic isoenzymes and their sensitivity towards suramin

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2',3'-ATP-Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga. Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308----His and Ala 306----Glu in the adenine-binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogues, but probably prevent phosphoryl-group transfer. Both isoenzymes displayed an activity optimum at pH 6.0-9.0 similar to that for the enzyme of yeast. Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Ki app. = 8.0 microM) to Suramin than either the cPGK (Ki app. = 20 microM) or the enzymes from rabbit muscle (Ki app. = 55 microM), yeast (Ki app. = 167 microM) or S. platensis (Ki app. = 250 microM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.     

Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.     

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targeting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.     

The cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei. Kinetic properties and comparison with homologous enzymes

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5-10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.     

Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides.

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.     

Use of enzyme ratios for differentiating stocks of Trypanosoma vivax

Bloodstream forms of nine different Trypanosoma vivax stocks were compared by the enzyme ratios of selected enzyme systems. Analysis of the results differentiated the trypanosome stocks into three groups, thus suggesting that enzyme ratios of selected enzymes could be of practical use in demonstrating intraspecific differences in trypanosomes.     

Trypanosoma pifanoi n. sp. from Colombian Bats*

SYNOPSIS. A new large trypanosome was found in the blood of 19 Artibeus lituratus and 2 Phyllostomus hastatus bats. The monomorphic trypanosome resembles Trypanosoma megadermae in some respects, but differs from it in that it is larger and has a short flagellum, both extremities are very tapered, the kinetoplast is very close to a small nucleus and there is a greater distance between the kinetoplast and the posterior extremity of the body. In diphasic blood-agar cultures there is a great variety of odd multiplication forms not described from other trypanosome cultures, but some simulate T. cruzi. This trypanosome is not capable of infecting mice, tissue culture cells, Carollia perspicillata bats, or triatomids, but is able to infect A. lituratus bats. Culture forms of the trypanosome inoculated intra-coelomically are pathogenic for several species of triatomids, and multiply in the hemolymph of Rhodnius prolizus, often producing forms similar to crithidiae of T. rangeli. Culture forms of the trypanosome seem to have common antigens with T. cruzi. This new species is described as Trypanosoma pifanoi.     

Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase

Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.     

Cell adhesion in Trypanosoma: in vitro studies of the interaction of Trypanosoma vivax with immobilized organic dyes

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.     

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.     

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.     

Cell Adhesion in Trypanosoma: In Vitro Studies of the Interaction of Trypanosoma vivax with Immobilized Organic Dyes1

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex? Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.     

The fatty acids of Trypanosoma vivax

A study was carried out to determine the lipid composition of the blood-stream form of the African trypanosome. Trypanosoma vivax. Data from thin layer chromatography showed that the major polar lipids were lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diphosphatidylglycerol. The major neutral lipids were sterol, monoacylglycerol, diacylglycerol, free fatty acid and triacyglycerol. 16:0, 18:0, 18:1 and 18:2 constituted the major fatty acids of both the polar and neutral lipid fractions. The work constituted the first detailed study on the fatty acid composition of this African trypanosome.     

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.     

Characterization of Trypanosoma (Duttonella) vivax by isoenzyme analysis

Stocks derived from 10 different primary isolates of T. vivax were subjected to isoenzyme analysis for 34 enzymes by both isoelectric focusing in agarose and electrophoresis in starch gel. Trypanosomes were measured and their morphology examined for comparison with the biochemical data. Thirteen enzymes (14 zymograms) were selected to construct isoenzyme profiles. Nine different zymodemes were identified and only two stocks were identical. Both rodent infectivity and the production of the haemorrhagic syndrome could be correlated with the isoenzyme profiles.