Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

Bourguignon, S.C.; Mn, Meirelles; Pacheco, R.; De-Simone, Salvatore Giovanni

doi:10.1590/s0074-02761998000200017

Cited by 4 publications

(2 citation statements)

References 27 publications

(29 reference statements)

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“…From the chromatogram obtained for the markers (gel filtration standard # 151-1901 from Bio-Rad), we found that protein mass of the FoxTPI protein corresponds to the native dimers with a relative molecular weigth (MW) of 56 kDa ( Figure 4B), and which is in accordance with the MW expected from the amino acid sequence (27,000 × 2 = 54 kDa), whereas the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a unique band of around 27 kDa (Figure 3). This result is consistent with those reported for other TPIs, such as of Saccharomyces cerevisiae [44], and parasites as Entamoeba histolytica (EhTPI), Trypanosoma brucei (TbTPI), T. cruzy (TcTPI), and Encephalitozoon intestinalis (EiTPI), which were reported with a molecular mass around 27 kDa [45][46][47][48].…”

Section: Oligomeric Status Of the Foxtpi Proteinsupporting

confidence: 92%

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

et al. 2019

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Triosephosphate isomerase (TPI) is a glycolysis enzyme, which catalyzes the reversible isomerization between dihydroxyactetone-3-phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). In pathogenic organisms, TPI is essential to obtain the energy used to survive and infect. Fusarium oxisporum (Fox) is a fungus of biotechnological importance due to its pathogenicity in different organisms, that is why the relevance of also biochemically analyzing its TPI, being the first report of its kind in a Fusarium. Moreover, the kinetic characteristics or structural determinants related to its function remain unknown. Here, the Tpi gene from F. oxysporum was isolated, cloned, and overexpressed. The recombinant protein named FoxTPI was purified (97% purity) showing a molecular mass of 27 kDa, with optimal activity at pH 8.0 and and temperature of 37 °C. The values obtained for Km and Vmax using the substrate GAP were 0.47 ± 0.1 mM, and 5331 μmol min−1 mg−1, respectively. Furthemore, a protein structural modeling showed that FoxTPI has the classical topology of TPIs conserved in other organisms, including the catalytic residues conserved in the active site (Lys12, His94 and Glu164). Finally, when FoxTPI was analyzed with inhibitors, it was found that one of them inhibits its activity, which gives us the perspective of future studies and its potential use against this pathogen.

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Section: Oligomeric Status Of the Foxtpi Proteinsupporting

confidence: 92%

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

et al. 2019

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“…In this context, it is relevant that Bourgignon et al (1998), using a polyclonal rabbit against TcTIM, observed that TcTIM was in the cytosol; albeit their electron microscope techniques did not allow them to ascertain if the enzyme localized exclusively in the glycosomes. Nonetheless, and along this line, it is relevant that Taylor and Gutteridge (1987) studied the subcellular location of glycolytic enzymes in T. cruzi; they found by isopycnic centrifugation that TcTIM was indeed in the glycosomes; however, they also found that a significant portion of the enzyme was in the lighter fractions, including the soluble fraction.…”

Section: Discussionmentioning

confidence: 99%

A monoclonal antibody that inhibits Trypanosoma cruzi growth in vitro and its reaction with intracellular triosephosphate isomerase

et al. 2007

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In parasites of the order Kinetoplastida, such as Trypanosoma cruzi and Trypanosoma brucei, glycolysis is carried out by glycolytic enzymes in glycosomes. One of the glycolytic enzymes is triosephosphate isomerase (TIM), which in T. brucei is localized exclusively in glycosomes, whereas in T. cruzi, the localization of TIM has not been fully ascertained. In the present work, we made a monoclonal antibody (mAb 6-11G) against recombinant T. cruzi TIM (rTcTIM). Incubation of T. cruzi epimastigotes with the mAb inhibited parasite survival. Western blotting showed that the mAb recognized rTcTIM and a 27 kDa band in T. cruzi lysates that corresponded to TcTIM. Sera from patients with Chagas disease recognized rTcTIM and cross-reacted with human recombinant TIM. The cross reactivity between parasite and human TIM possibly contributes to the autoimmune pathogenesis of Chagas disease. Electron microscopy of T. cruzi epimastigotes with the mAb showed that TIM was located within glycosomes, in the cytoplasm, the nucleus, and the kinetoplast. Collectively, the data shed new light on T. cruzi TIM and opens perspectives for drug design.

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Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

Auer

Camoin

Courtot

et al. 2004

Molecular Reproduction Devel

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P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.

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Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

Abstract: The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1)

Cited by 4 publications

References 27 publications

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

A monoclonal antibody that inhibits Trypanosoma cruzi growth in vitro and its reaction with intracellular triosephosphate isomerase

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

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