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

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 tryps...

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Bibliographic Details
Published in:Veterinary parasitology 2012-07, Vol.187 (3-4), p.521-528
Main Authors: Soares, Tatiane Sanches, Watanabe, Renata Midori Okuta, Tanaka-Azevedo, Anita Mitico, Torquato, Ricardo José Soares, Lu, Stephen, Figueiredo, Ana Carvalho, Pereira, Pedro José Barbosa, Tanaka, Aparecida S.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Anticoagulant
Antigens - immunology
Arthropod Proteins - genetics
Arthropod Proteins - metabolism
Arthropod Proteins - pharmacology
Boophilus microplus
Cloning, Molecular
Elastase inhibitor
Gastrointestinal Tract - metabolism
Gene Expression Regulation - physiology
Ixodidae
Kunitz-type inhibitor
Molecular Sequence Data
Pichia pastoris
Rhipicephalus
Rhipicephalus - metabolism
RNA Interference
RNAi silencing
Thrombin - antagonists & inhibitors
Tick
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Summary: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 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (Ki 0.65nM), neutrophil elastase (Ki 21nM) and bovine thrombin (Ki 57pM), while D1 inhibited trypsin and neutrophil elastase (Ki of 2.0 and 129nM, 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.
ISSN:0304-4017
1873-2550
DOI:10.1016/j.vetpar.2012.01.027