Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

Author Buarque, Diego de Souza Autor UNIFESP Google Scholar
Spindola, Leticia Maria Nery Autor UNIFESP Google Scholar
Martins, Rafael M. Google Scholar
Braz, Gloria R. C. Google Scholar
Tanaka, Aparecida Sadae Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
Inst Pasteur
Abstract The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K-i = 3.29 nM) and human cathepsin L (K-i= 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan. (C) 2011 Elsevier Inc. All rights reserved.
Keywords Cruzipain
Cysteine protease inhibitor
Tigutcystatin
Triatoma infestans
Trypanosoma cruzi
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
INCT - Entomologia Molecular
Grant number FAPESP: 05/03514-9
FAPESP: 07/58973-3
Date 2011-09-23
Published in Biochemical and Biophysical Research Communications. San Diego: Academic Press Inc Elsevier Science, v. 413, n. 2, p. 241-247, 2011.
ISSN 0006-291X (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 241-247
Origin http://dx.doi.org/10.1016/j.bbrc.2011.08.078
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000295443400016
URI http://repositorio.unifesp.br/handle/11600/34055

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