Sequencing and heterologous expression of a cytosolic-type malate dehydrogenase of Trypanosoma brucei.
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Amino Acid Sequence↗
Biomedical subjects
J J Cazzulo
Publications and source records attributed to J J Cazzulo.
The high stability of cruzipain against pH-induced inactivation is not dependent on its C-terminal domain.
Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) cruzipains exhibit similar stabilities at both acid (k(inac)=3.1x10(-3) s(-1) and 4.4x10(-3) s(-1) at pH 2.75 for endogenous and recombinant cruzipain, respectively) and alkaline pH (k(inac)=3.0x10(-3) s(-1) and 3. 7x10(-3) s(-1) at pH 9.15 for endogenous and recombinant cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.
Animals↗
Tetrameric and dimeric malate dehydrogenase isoenzymes in Trypanosoma cruzi epimastigotes.
Two malate dehydrogenase isoforms, named MDH1 and MDH2, have been purified to homogeneity from Trypanosoma cruzi epimastigotes. Both enzymes consist of subunits with a molecular mass close to 33 kDa; native molecular mass determination by gel filtration, however, indicated that MDH1 is a dimer, whereas MDH2 is a tetramer. Both isoforms did not cross-react immunologically. The N-termini of both MDH isoforms and several tryptic peptides of MDH1 (amounting to about one third of the complete molecule) have been sequenced by automated Edman degradation. The tryptic digests of both enzymes have also been analysed by mass spectrometry (MALDI-TOF MS). The apparent Km values in both directions of the reaction have been determined, as well as the possible inhibition by excess of the substrate oxaloacetate. The sequence data, together with the pI values and the presence or absence of oxaloacetate inhibition indicate that the dimeric MDH1 is the mitochondrial isoenzyme, whereas the tetrameric MDH2 is the glycosomal isoenzyme. No evidence was found for the presence of a cytosolic isoform.
Amino Acid Sequence↗
Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi.
A single form of serine hydroxymethyltransferase (SHMT) was detected in epimastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the enzyme characterized from another trypanosomatid, Crithidia fasciculata [Capelluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol. Biochem. Parasitol. 98, 187-201]. The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N-alpha-p-tosyl-L-lysine chloromethyl ketone and Ltrans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, however, the enzyme could be purified to homogeneity. Digitonin treatment of intact cells suggested that the enzyme is cytosolic. T. cruzi SHMT presents a monomeric structure shown by the apparent molecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephadex G-200 gel filtration and SDS/PAGE, respectively. This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate-dependent after L-cysteine and hydroxylamine treatments and it was strongly inhibited by the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards L-serine. Partial sequencing of tryptic internal peptides of the enzyme indicate considerable similarity with other SHMTs, particularly from those of plant origin.
Amino Acid Sequence↗
Purification and partial characterization of three isoforms of serine hydroxymethyltransferase from Crithidia fasciculata.
Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in choanomastigotes of Crithidia fasciculata by DEAE-cellulose chromatography. The three isoforms (named SHMT I, II, and III) presented small differences in charge and molecular weight. Digitonin treatment of intact cells suggested that SHMT III is cytosolic, whereas the other two isoforms are particle bound, one being mitochondrial (SHMT I) and the other one very likely glycosomal (SHMT II). The three SHMT isoforms were purified to homogeneity, and their physicochemical and kinetic properties studied. Determination of their native and subunit molecular masses revealed that all of them have a tetrameric structure. The three isoforms were shown to be PLP-dependent enzymes after L-cysteine and hydroxylamine hydrochloride treatments. They showed similar pH optima, bimodal kinetics for L-serine and Michaelis-Menten kinetics for THF.
Animals↗
The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi. A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity.
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
Alcohol Oxidoreductases↗
Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins.
Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.
1-Deoxynojirimycin↗
Cathepsin S and cruzipain are inhibited by equistatin from Actinia equina.
Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24 kDa and an isoelectric point in the range of 8.2 to 8.6. The enzyme is inhibited by equistatin, which belongs to the thyropins, a new family of protein inhibitors, with an inhibition constant of Ki = 0.40 +/- 0.07 nM. Cruzipain, a cathepsin L-like enzyme sharing a 130 amino acid long C-terminal extension, is also strongly inhibited by equistatin (Ki = 0.028 +/- 0.006 nM). Together with previously reported data, these results further indicate that a functional heterogeneity exists among thyropin inhibitors, as demonstrated by their interaction with cathepsin S and cruzipain.
Animals↗
[Cruzipain, major cysteine proteinase of Trypanosoma cruzi: sequence and genomic organization of the codifying genes].
Cruzipain is the major cysteine proteinase present in Trypanosoma cruzi, the parasite causing the American Trypanosomiasis, Chagas disease. The enzyme is encoded by a high number of genes (up to 130 in the Tul 2 stock) placed in head-to-tail tandems, and located in two to four chromosomes. The simultaneous expression of several different genes results in the production of a complex mixture of isoforms. Those known as a group as "cruzipain 1" differ essentially at the level of the C-terminal domain, in their amino-acid sequence and in their type of N-glycosylation. The existence of a more distantly related cysteine proteinase, "cruzipain 2", has been demonstrated; it differs markedly, particularly at the level of the catalytic moiety.
Amino Acid Sequence↗
Isolation and partial characterization of a broad specificity aminotransferase from Leishmania mexicana promastigotes.
A broad specificity aminotransferase (BSAT), with high activity with both, aromatic amino acids and aspartate as substrates, was purified to homogeneity from promastigotes of Leishmania mexicana by a method involving chromatography on DEAE-cellulose, Red-120-Sepharose and Mono Q, and gel filtration on Sephacryl S-200. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, was 90 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequence of four internal peptides obtained by tryptic digestion. Two of these peptides, consisting of 49 amino acid residues in total, showed high similarity (57%) with corresponding sequences of plant aspartate aminotransferases, whereas they had only 33% identity with the aromatic aminotransferase of Escherichia coli, and 16% identity with the tyrosine aminotransferase from the related parasite Trypanosoma cruzi. The BSAT contained only one 1/2 Cys residue per monomer. The optimal pH for the enzyme reaction, with tyrosine and alpha-oxoglutarate as substrates, was 7.0. The apparent Km values for tyrosine, phenylalanine, tryptophan and glutamate, with oxaloacetate as co-substrate, were 1.3, 0.9, 0.9 and 171.8 mM, respectively; the value for aspartate with alpha-oxoglutarate as co-substrate was 2.5 mM, and that for alanine with alpha-oxoglutarate as co-substrate was 216 mM. The values for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as co-substrate, were 5.6, 0.71 and 0.12 mM, respectively. These results suggest that the enzyme is a broad-specificity aminotransferase, able to transaminate the aromatic amino acids, aspartate, and to a lower extent alanine, with high sequence similarity to aspartate aminotransferases.
Amino Acid Sequence↗
Substrate inhibition of cruzipain is not affected by the C-terminal domain.
Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C-terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25 degrees C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.
Animals↗
The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression.
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
Amino Acid Sequence↗
Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain.
Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.
Animals↗
Membrane-bound cysteine proteinase isoforms in different developmental stages of Trypanosoma cruzi.
Cysteine proteinase isoforms, immunologically cross-reactive with cruzipain and with a similar apparent molecular mass, have been identified in epimastigotes of Trypanosoma cruzi by extraction and phase partition using the detergent Triton X-114. These isoforms are concentrated in the microsomal fraction obtained after differential centrifugation, which is known to consist essentially of plasma membrane, can be labelled by incubation of live parasites with sulfo-NHS-biotin, and bind to cystatin-sepharose affinity columns. They are present, albeit with a different electrophoretic pattern, in the epimastigote, amastigote and trypomastigote stages of the parasite.
Animals↗
Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi.
SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 microM concentrations. When blood from infected mice was treated with the drug, and used to infect 8 day-old mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks.
Animals↗
A fragment of the major histocompatibility complex class II-associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi.
A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k(ass) = 2.4 x 10(7) M(-1) s(-1) and tight (Ki = 5.8 x 10(-11) M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.
Alternative Splicing↗
Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi.
Trypanosoma cruzi, the parasitic protozoan which causes the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain. The enzyme belongs to the papain family, but contains, as other CPs from Trypanosomatids, an unusual C-terminal extension. This C-terminal domain contains a number of post-translational modifications and is responsible for the immunodominant antigenic character of cruzipain in natural human infections. In addition, this domain is probably the cause of most of the microheterogeneities found in natural cruzipain. Irreversible inhibitors of CPs are able to block the parasite's life cycle at the differentiation steps, suggesting an essential role for CPs for parasite survival, and opening up possibilities of developing new chemotherapeutic agents against Chagas disease based on specific cruzipain inhibitors.
Amino Acid Sequence↗
High-molecular-weight kininogen binds two molecules of cysteine proteinases with different rate constants.
Fluorescence titrations showed that high-molecular-weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kass,1 = 23.0 x 10(6) M-1 s-1 and kass,2 = 3.4 x 10(6) M-1 s-1). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low- and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M(r) of high-molecular-weight kininogen was determined to be 83,500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.
Animals↗