QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Monteiro, A. C. S. Articles by Scharfstein, J. Search for Related Content PubMed PubMed Citation Articles by Monteiro, A. C. S. Articles by Scharfstein, J. Social Bookmarking                         What's this?

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi

¹ Instituto de Biofísica Carlos Chagas Filho, Bloco G, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, CEP 21990-400, Brazil
² Department of Clinical Chemistry, University of Lund, University Hospital, S-221 85 Lund, Sweden

View larger version (23K): [in a new window]   Fig. 1. Detection of a cruzipain binding protein in T. cruzi epimastigotes lysates. Proteins from epimastigotes lysates, or recombinant human cystatin C used as a control, were diluted in SDS-sample buffer (without boiling) under non-reducing conditions. After running a 16% SDS-PAGE, the polypeptides were transferred to nitrocellulose membranes. Proteins that were capable of binding to cysteine proteases were identified by incubating the membranes with (A) cruzipain (10 µg/ml) in PBS, 2 mM EDTA, 1% BSA (w/v), 0.05% (v/v) Tween 20 for 1 hour at room temperature. After washing, the bound cruzipain probe was identified by treating the membrane with mAbcruzipain. Peroxidase-labeled anti-mouse IgG was used to visualize the immuno-reactive bands (B), membrane treated with buffer (instead of the cruzipain probe) followed by incubation with mAbcruzipain. Lane 1: recombinant human cystatin C (10 ng/ml); lane 2: epimastigote lysate.

View larger version (64K): [in a new window]   Fig. 2. Purification of n-chagasin. (A) Silver staining of natural chagasin (arrow) purified from epimastigote lysates as described in Materials and Methods. (B) Immunoblotting of the purified protein, carried out with a rabbit antiserum directed against r-chagasin. Electrophoresis run on 16% SDS-PAGE under denaturing conditions.

View larger version (45K): [in a new window]   Fig. 3. Expression of chagasin during the life cycle of T. cruzi. Western blotting of Triton X-100 lysates (10 µg protein) from the three developmental stages of Dm28c T. cruzi. The membranes were incubated with rabchagasin (1:1000) in PBS, 2 mM EDTA, 3% (w/v) nonfat dried milk, 0.05% (v/v) Tween 20 for 1 hour at room temperature. Lane 1, r-chagasin as a reference (100 ng); lane 2, trypomastigotes; lane 3, epimastigotes; and lane 4, amastigotes.

View larger version (77K): [in a new window]   Fig. 4. Expression of chagasin RNA in T. cruzi. Total RNA (20 µg) from trypomastigotes (lane 1), epimastigotes (lane 2) or amastigotes (lane 3) were separated in a 2% agarose-formaldehyde gel, transferred onto nylon membranes and probed with [-32P]chagasin cDNA in 5x SSC, 5x Denhardt’s, 1% SDS, 50 µg/ml salmon sperm DNA, 50% formamide at 42°C, overnight, washed three times with 0.2xSSPE, 0.1% SDS, and exposed overnight at –70°C. Left panel: Ethidium bromide staining of the gel. Right panel: Autoradiograph. The approximate position of co-eletrophoresed RNA molecular weight marker is indicated on the right.

View larger version (8K): [in a new window]   Fig. 5. Screening of anti-chagasin antibodies in human sera. A panel of 25 sera from chronic chagasic patients, and 25 normal control human sera, were tested at 1:50 dilution against immobilized r-chagasin by ELISA. Following incubation with anti-human IgG conjugated to peroxidase, the color development was detected at 492 nm. Control wells coated with BSA and exposed to serum samples did not yield reactions (not shown).

View larger version (12K): [in a new window]   Fig. 6. Phase partition of n-chagasin following Triton X-114 extraction. Cells representing the three developmental stages of T. cruzi (epimastigotes, amastigotes and trypomastigotes) were lysed in 100 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.2% of pre-condensed Triton X-114, 0.1% (w/v) NaN3, 5 mM benzamidinium chloride and incubated with GPI-PLC (+) or not (-), for 30 minutes at 30°C. After subjecting these lysates to phase partition in Triton X-114, the samples were run on 15% SDS-PAGE and analyzed by western blotting, using rabchagasin serum. A, aqueous phase; D, detergent phase.

View larger version (138K): [in a new window]   Fig. 7. Sub-cellular localization of chagasin. Trypomastigotes (A,B) displayed intense and moderate labeling in flagellar pocket (A) and cytoplasmic vesicles (B, arrow), respectively, but cell surface was poorly labeled (arrowheads). Intermediate forms (C) presented intensely labeled cell surface (arrowheads) as well as flagellar pocket. T. cruzi amastigote (D) showing intense immunolabeling at both cell surface (arrowheads) and flagellar pocket. Flagellar pocket (P), flagellum (F), kinetoplast (K) and nucleus (N).

View larger version (78K): [in a new window]   Fig. 8. Chagasin and cruzipain bind to the cell surface of T. cruzi amastigotes and trypomastigotes. Living parasites were washed twice in PBS, 2 mM EDTA, incubated in the same buffer containing FITC-chagasin or FITC-cruzipain, for 30 minutes at 4°C, washed and fixed with 3% paraformaldehyde. Upper panels are phase-contrast and the lower ones are fluorescence microscopy. (A,B) Cell surface binding of FITC-chagasin (E,F) Cell surface binding of FITC-chagasin preincubated with unlabeled n-cruzipain. (C,D) Cell surface binding of FITC-cruzipain.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter