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J J Cazzulo

Publications and source records attributed to J J Cazzulo.

At least 55 records · Page 3Linked to original sources
PubMed1994

Production and characterization of monoclonal antibodies against the major cysteine proteinase of Trypanosoma cruzi.

In the present study we describe the production and characterization of a panel of monoclonal antibodies (MoAbs) directed against cruzipain (Crz), the major cysteine proteinase from Trypanosoma cruzi. The five MoAbs, BD6, BF2, CG2, CH8, and DC10 were analysed with respect to affinity and specificity. None of the MoAbs cross-reacted with papain, which has regions of high homology with Crz. Treatment of the antigen with periodate did not affect the binding of the MoAbs, suggesting that they bind to the polypeptide moiety of Crz. CH8 recognized a continuous epitope located at the C-terminal extension of the proteinase that appeared to be highly immunogenic. Although the rest of the MoAbs recognized epitopes located in the catalytic domain, the enzymatic activity of Crz was not impaired by the binding of the MoAbs. Characterization of the antibody-binding sites revealed the presence of at least four separate epitopes.

Animals↗
PubMed1993

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi.

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

Amino Acid Sequence↗
PubMed1993

The reactivity of sera from chagasic patients against different fragments of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, suggests the presence of defined antigenic and catalytic domains.

Three fragments of cruzipain, expressed separately in bacterial vectors, were used to detect antibodies in sera from patients with chronic Chagas' disease. Most antibodies directed against the enzyme were found to react with the C-terminal extension, thus suggesting the presence of immunodominant B-cell epitopes within this protein domain. Immunoprecipitation with these antibodies did not impair enzyme activity. It is suggested that cruzipain consists of an enzymatic domain and a non-enzymatic, immunodominant domain, which corresponds to the C-terminal extension.

Animals↗
PubMed1993

Identity of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi and an antigen (Ag163B6) isolated with a monoclonal antibody.

Cruzipain, purified by conventional methods, and Ag163B6, isolated by affinity chromatography with a monoclonal antibody raised against a T. cruzi extract, are glycoproteins with a similar electrophoretic mobility, which reacted with sera from most chronic chagasic patients. Their behaviour in SDS-PAGE, Western blotting, isoelectric focusing, two-dimensional electrophoresis (IEF and SDS-PAGE), Ouchterlony's double diffusion, and enzyme activity in SDS-PAGE gels containing 0.1% gelatin suggests that they are identical.

Animals↗
PubMed1993

Purification of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi by affinity chromatography.

The major cysteine proteinase from Trypanosoma cruzi, cruzipain, can be obtained essentially homogeneous, starting from crude epimastigote extracts, in one step, by affinity chromatography on Cystatin-Sepharose (specific for cysteine proteinases) or ConA-Sepharose (specific for high mannose N-linked glycoproteins). The methods offer considerable potential for enzyme purification from scarce sources, such as other parasite stages or radioactively labelled material with high specific radioactivity.

Animals↗
PubMed1992

On the post-translational modifications at the C-terminal domain of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, has a 130 amino acid-long C-terminal domain, which, although microheterogeneous in SDS-PAGE, has a single N-terminal amino acid sequence. Most of the Thr residues present at the beginning of this sequence are modified; the nature of this modification is still unknown, but O-glycosylation and phosphorylation seem both to be absent. The only potential site for N-glycosylation (Asn 254) is glycosylated in vivo. Most of the eight Cys residues are involved in disulfide bridges. The results are consistent with cruzipain being made of two well-defined domains, a catalytic one with high homology to cathepsin L, and a C-terminal domain, linked to the former by a 'hinge' corresponding to the Pro- and Thr-rich region at its N-terminus.

Amino Acid Sequence↗
PubMed1992

Anomalous electrophoretic behaviour of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi in relation to its apparent molecular mass.

The molecular mass of cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, is 36.3 kDa as calculated from its sequence; this value can increase to about 41 kDa if the three potential N-glycosylation sites are glycosylated in vivo. Yet the apparent molecular mass of the enzyme, as determined by SDS-polyacrylamide gel electrophoresis, has been reported in a range of values from 60 to 40 kDa. We show that the purified enzyme had apparent molecular masses ranging from 51 to 33 kDa, depending on the experimental conditions. This variation is likely to be due to both N-glycosylation and the presence of several disulfide bridges, which make electrophoretic mobility dependent on acrylamide concentration, and reduction and/or boiling of the sample.

Animals↗
PubMed1992

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi.

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

Animals↗
PubMed1992

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes.

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Amino Acid Sequence↗
PubMed1992

A superfamily of Trypanosoma cruzi surface antigens.

Several genes o f Trypanosoma cruzi encode surface antigens that include an amino acid motif that is conserved among bacterial neurominidases. Oscar Campetella, Daniel Sdnchez, Juan Jose Cazzulo and Alberto Carlos Frasch here suggest grouping these gene families in a superfamily.

Journal Article↗
PubMed1992

Aerobic fermentation of glucose by trypanosomatids.

The consumption of glucose by trypanosomatid protozoa such as Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp., and Crithidia spp. is characterized by the excretion of reduced products such as succinate, pyruvate, ethanol, L-alanine, or lactate (depending on the species) not only in anaerobiosis, but also under aerobic conditions. The "aerobic fermentation" of glucose is accompanied by a complete lack, or even a reversal, of the Pasteur effect. This peculiar catabolism is mediated by a so-far unique compartmentation of the glycolytic enzymes, most of which are placed in an organelle called the glycosome; by an almost complete lack of inhibitory controls at the level of hexokinase and phosphofructokinase; and by a central role of CO2 fixation through the reaction catalyzed by phosphoenolpyruvate carboxykinase. The production of fermentative products seems to be due to a relative inefficiency of the respiratory chain, which lacks NADH dehydrogenase and the first phosphorylation site and preferentially uses succinate as substrate.

Aerobiosis↗
PubMed1992

SAPA/trans-sialidase and cruzipain: two antigens from Trypanosoma cruzi contain immunodominant but enzymatically inactive domains.

Trypanosoma cruzi, the parasitic protozoan that causes the American trypanosomiasis, or Chagas disease, contains a number of antigenic molecules, some of which have tandems of amino acid repeats. One of these molecules, SAPA (shed acute phase antigen), contains a so-far unique trans-sialidase activity that is essential for penetration of the parasite into mammalian cells. The enzyme consists of two different domains, one presumably enzymatic, which contains four copies of an amino acid motif conserved in bacterial neuraminidases, and the other highly antigenic, consisting of the repeats. Another enzyme that seems to be involved in the host-parasite relationship, the cysteine proteinase cruzipain, is also made up in its mature form of a catalytic domain with high homology to cathepsin L and a COOH-terminal domain that is highly antigenic in vivo. These bifunctional molecules may have arisen by incorporation of a highly antigenic domain to an essential enzyme in order to attract the immune response, thus protecting the enzyme activity.

Amino Acid Sequence↗
PubMed1991

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes of Trypanosoma cruzi.

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of L-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biochem. 192 (1990) 363-368).

Alanine Transaminase↗
PubMed1991

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Adenosine Triphosphatases↗
PubMed1991

A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum.

N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.

1-Deoxynojirimycin↗