Purification and partial structural and kinetic characterization of an alanine aminotransferase from epimastigotes of Trypanosoma cruzi.
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Alanine Transaminase↗
Biomedical subjects
J J Cazzulo
Publications and source records attributed to J J Cazzulo.
The use of UDP-Glc:glycoprotein glucosyltransferase for radiolabeling protein-linked high mannose-type oligosaccharides.
A simple method for specifically radiolabeling high mannose-type oligosaccharides linked to protein backbones has been developed. The method is based on the fact that incubation of rat liver UDP-Glc:glycoprotein glucosyltransferase, glucose-labelled UDP-Glc and a denatured high mannose-type glycoprotein target leads to the glucosylation of the oligosaccharide. In the case described here it allowed to follow easily the purification, by HPLC and affinity chromatography, of labelled glycopeptides obtained by controlled proteolysis of cruzipain, a cysteine proteinase isolated from the human pathogen Trypanosoma cruzi. It was thus determined that the N-glycosylation site in Asn33 of cruzipain is occupied by high mannose-type oligosaccharides.
Amino Acid Sequence↗
Hydrolysis of synthetic peptides by cruzipain, the major cysteine proteinase from Trypanosoma cruzi, provides evidence for self-processing and the possibility of more specific substrates for the enzyme.
Two synthetic peptides, peptide G, with the sequence KEEASSAVVGGPG, consisting of the last 10 amino acid residues of the catalytic domain, plus the first 3 at the C-terminal domain, of cruzipain, and peptide R, with the sequence KEEASSAVVRGPG, were hydrolyzed by the enzyme, as shown by reversed-phase HPLC. Peptide R was the best substrate, with a Vmax/K(m) ratio 6-fold higher as compared with peptide G, in good agreement with previous studies indicating that, in small peptides, cruzipain prefers R or K at P1. The optimal pH values for hydrolysis of peptides G and R were 6.8 and 8.0, respectively. A p-nitroanilide derivative containing the P1-P3 residues, Z-VVR-pNA, was an excellent substrate for cruzipain, with a K(m) value (33 microM at pH 9.0) lower than that for Bz-PFR-pNA (66 microM). These results open the possibility of synthesizing more specific substrates and inhibitors of cruzipain.
Amino Acid Sequence↗
A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities.
Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-transferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.
Alanine Transaminase↗
Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily.
Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5-9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (kass = 1.7-79 x 10(6) M-1s-1, Kd = 1.4-72 pM). These results suggest a possible defensive role for the host's cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.
Animals↗
Genes for cysteine proteinases from Trypanosoma rangeli.
PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli, using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi, allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.
Amino Acid Sequence↗
Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli.
Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.
Amino Acid Sequence↗
The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain.
Cruzipain is a lysosomal enzyme of the flagellate Trypanosoma cruzi. It has three potential asparagine-glycosylation sites, two in the catalytic domain and one in the C-terminal domain. The latter appeared to have both high mannose- and complex-type oligosaccharides, whereas the catalytic domain only had compounds of the former type. The partial susceptibility of the complex-type compounds to endo-beta-N-acetylglucosaminidase H and their relative mannose and galactose content indicate that they had hybrid/monoantennary and biantennary structures. The same pattern of high mannose-type compounds was found at both domains, thus indicating that in cruzipain molecules having only high mannose-type compounds, all oligosaccharides were equally exposed to processing glycosidases and glycosyltransferases. As heterogenity of the protein C-terminal domain has already been detected, it is suggested that this feature might elicit an increased accessibility to processing enzymes responsible for complex-type oligosaccharide formation in certain cruzipain molecules or, alternatively, that a second glycosylation site with increased accessibility might be present in certain cruzipain molecules. Furthermore, the presence of complex-type oligosaccharides strongly suggests that, as in mammalian cells, T. cruzi lysosomal enzymes traverse the entire Golgi apparatus up to the trans-Golgi cisternae and the trans-Golgi network before reaching lysosomes.
Animals↗
Purification and partial characterization of the cytosolic malate dehydrogenase from protoscolices of Echinococcus granulosus.
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Amino Acid Sequence↗
Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi.
The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.
Animals↗
Phosphoenolpyruvate carboxykinase from Trypanosoma cruzi. Purification and physicochemical and kinetic properties.
Phospho enolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of the Tul 0 strain of Trypanosoma cruzi. The physicochemical parameters determined allowed the calculation of an average molecular mass of 120 kDa; the subunit molecular mass, about 61 kDa, is in good agreement with the value of 58.6 kDa recently determined from the sequence by Sommer et al. (FEBS Lett. 359 (1994) 125-129). The PEPCK from T. cruzi presented, in addition to its molecular mass, typical properties of other ATP-linked PEPCKs, namely strict specificity for ADP in the carboxylation reaction and lower specificity in the decarboxylation and exchange reactions, and synergistic activation by CdCl2 or MgCl2 when added in addition to MnCl2. The enzyme presented hysteretic behaviour, shown by a lag period in the carboxylation reaction, which was affected by dilution and preincubation. The decarboxylation reaction catalyzed by the T. cruzi PEPCK was not inhibited by excess of ATP-Mn. The apparent Km values for the carboxylation reaction, including the low value for PEP (0.035 mM) are compatible with an important role of PEPCK, as suggested by previous NMR experiments, on the CO2 fixation in vivo which leads to succinate excretion during aerobic fermentation of glucose.
Animals↗
Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.
It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.
1-Deoxynojirimycin↗
Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola.
The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.
Adenosine Triphosphate↗
Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera.
Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same antisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.
Animals↗
Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.
Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.
Animals↗
Production of aromatic alpha-hydroxyacids by epimastigotes of Trypanosoma cruzi, and its possible role in NADH reoxidation.
Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p-hydroxyphenyllactic acids, presumably arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic alpha-hydroxyacid dehydrogenase.
Animals↗
Intermediate metabolism in Trypanosoma cruzi.
Epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of MH3, and glucose with production of reduced catabolites, chiefly succinate and L-alanine, even under aerobic conditions. This "aerobic fermentation of glucose" is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH, reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate or L-alanine, and L-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic alpha-hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.
Aerobiosis↗
Purification and partial structural and kinetic characterization of an aromatic L-alpha-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi.
An aromatic L-alpha-hydroxyacid dehydrogenase (AHADH) was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl-Sepharose and affinity chromatography on Affi-Gel Blue. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is about 80 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequences of 4 internal peptides obtained by CNBr cleavage at Met residues, and one peptide obtained after tryptic digestion. Three of the peptides presented considerable sequence similarity with the corresponding sequences of several malate dehydrogenases. The optimal pH for the enzyme reaction with p-hydroxyphenyl pyruvate and NADH as substrates was 7.5; that for the reverse reaction was 9.5. The apparent Km values for phenylpyruvate and p-hydroxyphenyl-pyruvate were 48 and 117 microM, respectively; that for L-phenyllactate in the reverse reaction was 420 microM. The enzyme was much less active with alpha-isocaproic acid as substrate, and other acids, including pyruvic and oxaloacetic, were not substrates at all. L-phenyllactic acid, but not the D-isomer, acted as substrate. The enzyme can therefore be considered as a general aromatic L-alpha-hydroxyacid dehydrogenase. The low apparent Km value for NADH (25 microM in the presence of phenylpyruvate) makes AHADH a candidate for the reoxidation of cytosolic NADH in T. cruzi.
Alcohol Oxidoreductases↗