Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.     

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.     

Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 A resolution

The three-dimensional crystal structure of the enzyme triosephosphate isomerase from the unicellular tropical blood parasite Trypanosoma brucei brucei has been determined at 2.4 A resolution. This triosephosphate isomerase is sequestered in the glycosome, a unique trypanosomal microbody of vital importance for the energy-generating machinery of the trypanosome. The crystals contain one dimer per asymmetric unit. The structure could be solved by the method of molecular replacement, using the refined co-ordinates of chicken triosephosphate isomerase as a search model. The positions and individual isotropic temperature factors of the 3792 atoms of the complete dimer have been refined by the Hendrickson & Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances, the R-factor has dropped to 23.2% for 12317 reflections between 6 A and 2.4 A. A total of 0.6 mg of enzyme was used for establishing the correct crystallization conditions and solving the three-dimensional structure. Although the sequences of trypanosomal and chicken triosephosphate isomerase are identical at only 52% of the 247 common positions, the overall folds are very similar. The architecture of the active sites is virtually the same with 85% of the side-chains being identical. On the other hand, the residues involved in the dimer contacts are the same at only 55% of the positions. Nevertheless, the position of the local 2-fold axis in the chicken and glycosomal dimers is similar. A remarkable feature of glycosomal triosephosphate isomerase is its high overall positive charge. This extra charge is concentrated in four clusters of positively charged side-chains on the surface of the dimer, quite far away from the active site. These clusters may be involved in the mechanism of import of this triosephosphate isomerase into the glycosome.     

Preliminary crystallographic studies of glycosomal glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei

Crystals of glyceraldehyde phosphate dehydrogenase from the glycosome of Trypanosoma brucei brucei have been grown, and a partial data set has been collected using synchrotron radiation. The crystals diffract initially to 2.3 A resolution. The space group is P2(1)2(1)2, with cell dimensions a = 135 A, b = 255 A, c = 115 A, so there are probably at least two tetramers in the asymmetric unit.     

The 3.0 A resolution crystal structure of glycosomal pyruvate phosphate dikinase from Trypanosoma brucei

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement. The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.     

Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.

The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.     

Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei

Background

Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.

Methods/Principal Findings

We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P _K+/P _Cl−∼0.31), while the other two types of channels are slightly selective for cations (P _K+/P _Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel''s pore.

Conclusions/Significance

These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.     

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.     

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde-phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody-like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52-57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1-8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.     

Biogenesis of the glycosome in Trypanosoma brucei: the synthesis, translocation and turnover of glycosomal polypeptides.

Glycosomes, the microbodies of Trypanosoma brucei, contain a number of enzymes involved in glucose and glycerol metabolism. The biogenesis of three of these enzymes has been studied. Aldolase, D-glyceraldehyde-3-phosphate dehydrogenase and NAD-linked glycerol-3-phosphate dehydrogenase are all synthesized in the cytosol on free rather than on membrane-bound polysomes. In vitro, as well as in vivo, these polypeptides are synthesized at their mature size, and no evidence was found for any processing upon entry into the glycosomes. Continuous and pulse-chase labelling experiments with procyclic trypomastigotes revealed that the enzymes have a half-life in the cytosol of approximately 3 min or less, and then turn over rapidly in the glycosomes, with half-lives as short as 30 min.     

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins.

It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with glycosomes, the peroxisome-like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N-terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome-targeting signal type 1 (PTS-1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream-form and procyclic, insect-stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1, a N-terminal PTS-2 and a polypeptide internal I-PTS. The RNA interference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypanosomes. These data indicate the protein's great potential as a target for selective trypanocidal drugs.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: Inhibition by Organotins

Activity and kinetics of phospholipase A₂ (PLA₂) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca²⁺-independent, strongly stimulated by Triton X-100 with optimum activity at 37°C and pH 6.5–8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA₂. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (K_M) for PLA₂ from T. b. brucei is 63.87 and 30.90 μM while for T. b. gambiense it is 119.64 and 32.90 μM for the substrates l,2-bis-(1-pyrenebutanoyl-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dode-canoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC), respectively.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei

Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein. We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes. Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C. This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals. The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli. Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C. These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T. brucei.     

The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.