Detection of peptidases in<i>Trypanosoma cruzi</i>epimastigotes using chromogenic and fluorogenic substrates

Healy, Niamh; Greig, Stephen; Enahoro, H.; Roberts, H C; Drake, Lynn A.; Shaw, Elliott; Ashall, Frank

doi:10.1017/s003118200006176x

Cited by 13 publications

(7 citation statements)

References 23 publications

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“…Synthetic peptide substrates containing AMC, MNA or 3-amino-o-ethylcarbazole fluorescent groups have been used as highly sensitive and specific tools to study proteinases in several species of protozoa [40]. Some T. cruzi proteolytic activities have been studied using such substrates [18,39,41,42]. In the present work, we demonstrate that cell-free extracts of T. cruzi amastigote, trypomastigote and epimastigote forms readily cleave a collagenase fluorogenic substrate with an optimal pH of about 8n0.…”

Section: Discussionmentioning

confidence: 57%

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV

Santana

Grellier

Schrével

et al. 1997

Biochemical Journal

132

105

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Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy.

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Section: Discussionmentioning

confidence: 57%

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV

Santana

Grellier

Schrével

et al. 1997

Biochemical Journal

132

105

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“…The plate was incubated at 30°C and read intermittently. Under these assay conditions, it was confirmed that the protease activity was sensitive to 40 M trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E64; results not shown) and therefore corresponded to the lysosomal cysteine proteinase cruzipain (29,30) and not the cytosolic alkaline protease that is insensitive to this inhibitor (31). The release of pnitrophenol or p-nitroaniline from substrates was measured using a Dynatech MR5000 reader at 410 -450 nm.…”

Section: )mentioning

confidence: 89%

In Situ Compositional Analysis of Acidocalcisomes in Trypanosoma cruzi

Scott

Docampo

Dvorak

et al. 1997

Journal of Biological Chemistry

108

129

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We measured the elemental content of different compartments in Trypanosoma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron density contained (in units of mmol/kg dry weight ؎ S.E.) large amounts of phosphorus (1390 ؎ 13), magnesium (646 ؎ 19), calcium (171 ؎ 5), sodium (161 ؎ 18), and zinc (148 ؎ 6). No other compartment had appreciable calcium or zinc content. Iron (128 ؎ 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3-or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in the other elements investigated. This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes, i.e. acidic compartments rich in Ca 2؉ . The acidocalcisomes were investigated by separation of epimastigote fractions on Percoll gradients in combination with Triton WR-1339 treatment. This detergent caused a rapid vacuolation; these vacuoles were shown by electron microscopy to be largely transparent, with a diffuse matrix. Percoll gradient fractionation demonstrated decreases in the density of various organelle markers in detergent-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as smaller decreases in the density of the other markers. Conventional electron microscopy of epimastigotes loaded with gold-labeled transferrin indicated that the endosomal system was separate from vacuoles that probably corresponded to the calcium-containing organelles detected by electron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites.

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“…Additionally, the cysteine protease cathepsin B, a serine carboxipeptidase, and, more recently, two cytosolic metallocarboxypeptidases, a serine oligopeptidase and two aspartyl proteases have been biochemically characterized [13-17]. In contrast, the study of aminopeptidases has been limited to the detection of such activity in cell extracts of T. cruzi epimastigotes [18]. …”

Section: Introductionmentioning

confidence: 99%

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

et al. 2011

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BackgroundPathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease.ResultsThe enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH.ConclusionsLAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.

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Detection of peptidases inTrypanosoma cruziepimastigotes using chromogenic and fluorogenic substrates

Cited by 13 publications

References 23 publications

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV

In Situ Compositional Analysis of Acidocalcisomes in Trypanosoma cruzi

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

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