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

Autor(es)

Buarque, Diego S. ^[1] ; Spindola, Leticia M. N. ^[1] ; Martins, Rafael M. ^[2] ; Braz, Gloria R. C. ^[3] ; Tanaka, Aparecida S. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 Sao Paulo - Brazil

^[2] Inst Pasteur, Biol Host Parasite Interact Unit, F-75015 Paris - France

^[3] Univ Fed Rio de Janeiro, Dept Biochem, Inst Quim, BR-21941909 Rio De Janeiro - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biochemical and Biophysical Research Communications; v. 413, n. 2, p. 241-247, SEP 23 2011.
• Citações Web of Science: 9

Resumo

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. (AU)

• Processo FAPESP: 05/03514-9 - Estudos da função fisiológica e potencial biotecnológico de inibidores de proteases e anti-hemostáticos presentes em artrópodes hematófagos
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 07/58973-3 - Estudo do perfil de genes e proteinas do intestino medio de triatoma infestans na presenca do trypanosoma cruzi.
• Beneficiário: Diego de Souza Buarque
• Modalidade de apoio: Bolsas no Brasil - Doutorado