Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases.
ABSTRACT The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.
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ABSTRACT: We have analyzed the proteinase profile of two Herpetomonas species, H. anglusteri and H. roitmani (a symbiont-bearing trypanosomatid), by in situ detection of enzyme activities on SDS-PAGE gels containing copolymerized gelatin as substrate. Two major cell-associated proteolytic activities, a 60 kDa zinc-metalloproteinase and a 45 kDa cysteine proteinase could be detected based on the inhibition of their activities by 1, 10-phenathroline and E-64, respectively. The trypanosomatids released into the growth medium distinct proteinases. H. anglusteri expressed three digestion haloes in the gels of approximately 60, 50, and 40 kDa, whereas H. roitmani secreted only a 60 kDa enzyme. However, these activities were inhibited by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinase.Current Microbiology 09/1999; 39(2):61-4. · 1.52 Impact Factor
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ABSTRACT: Peptidomimetic inhibitors of mammalian zinc metalloproteases have been tested as potential agents for intervention in disease caused by kinetoplastid protozoa. Certain metalloprotease inhibitors were able to inhibit the release of variant surface glycoprotein from cultured transgenic procyclic Trypanosoma brucei, confirming our previous identification of a cell surface zinc metalloprotease activity in this stage of the trypanosome lifecycle [Bangs, JD et al. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion, EMBO J. 1997;16:4285]. Selected peptidomimetics were also found to be toxic for cultured bloodstream trypanosomes with IC50 values in the low micromolar range. The paradigm for zinc metalloproteases in kinetoplastids are the GP63 surface enzymes of Leishmania. Peptidomimetics at low micromolar concentrations were able to inhibit in vitro cleavage of a synthetic peptide substrate by purified GP63 from L. major. Our results suggest that zinc metalloproteases perform essential functions in different stages of the trypanosome lifecycle and we hypothesize that these activities may be affected by the recently discovered trypanosomal homologues of GP63 [El-Sayed, NMA and Donelson, JE. African trypanosomes have differentially expressed genes encoding homologues of Leishmania GP63 surface protease, J. Biol. Chem. 1997;272:26742]. Development of higher affinity metalloprotease inhibitors may provide a novel avenue for treatment of parasitic diseases.Molecular and Biochemical Parasitology 05/2001; 114(1):111-7. · 2.73 Impact Factor
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ABSTRACT: Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin L and cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors of cruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasite's life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine-Phe-hPhe-vinyl sulphone phenyl) which is an efficient inhibitor of cruzipain and kills T. cruzi by inducing an accumulation of unprocessed cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.Current Pharmaceutical Design 09/2001; 7(12):1143-56. · 3.31 Impact Factor