Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Author Soares, Tatiane Sanches Autor UNIFESP Google Scholar
Watanabe, Renata Midori Okuta Autor UNIFESP Google Scholar
Tanaka-Azevedo, Anita Mitico Google Scholar
Torquato, Ricardo Jose Soares Autor UNIFESP Google Scholar
Lu, Stephen Autor UNIFESP Google Scholar
Figueiredo, Ana Carvalho Google Scholar
Pereira, Pedro Jose Barbosa Google Scholar
Tanaka, Aparecida Sadae Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Inst Butantan
Univ Porto
Abstract Rhipicephalus (Boophilus)microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. the large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5 mg/L of culture, respectively. Purified boophilin inhibited trypsin (K-i 0.65 nM), neutrophil elastase (K-i 21 nM) and bovine thrombin (K-i 57 pM), while D1 inhibited trypsin and neutrophil elastase (K-i of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control. (C) 2012 Elsevier B.V. All rights reserved.
Keywords Kunitz-type inhibitor
Tick
Anticoagulant
Elastase inhibitor
RNAi silencing
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
Fundacao para a Ciencia e a Tecnologia, Portugal
EU-FEDER
POCI
Grant number FAPESP: 05/03514-9
FAPESP: 09/05405-3
CNPq: 490574/2006-8
Fundacao para a Ciencia e a Tecnologia, Portugal: PTDC/BIA-PRO/70627/2006
Fundacao para a Ciencia e a Tecnologia, Portugal: REEQ/564/1310/2005
: SFR/BPD/46722/2008
Date 2012-07-06
Published in Veterinary Parasitology. Amsterdam: Elsevier B.V., v. 187, n. 3-4, p. 521-528, 2012.
ISSN 0304-4017 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 521-528
Origin http://dx.doi.org/10.1016/j.vetpar.2012.01.027
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000307323700024
URI http://repositorio.unifesp.br/handle/11600/35089

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