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

Author(s)

Soares, Tatiane Sanches ^[1] ; Okuta Watanabe, Renata Midori ^[1] ; Tanaka-Azevedo, Anita Mitico ^[2] ; Soares Torquato, Ricardo Jose ^[1] ; Lu, Stephen ^[1] ; Figueiredo, Ana Carvalho ^[3] ; Barbosa Pereira, Pedro Jose ^[3] ; Tanaka, Aparecida S. ^[1]

Affiliation

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

^[2] Inst Butantan, Lab Fisiopatol, Sao Paulo, SP - Brazil

^[3] Univ Porto, IBMC, P-9150180 Oporto - Portugal

• Document type: Journal article
• Source: Veterinary Parasitology; v. 187, n. 3-4, p. 521-528, JUL 6 2012.
• Web of Science Citations: 25

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. (AU)

• FAPESP's process: 09/05405-3 - Automatic DNA sequencer update of INFAR Multi-user Laboratory, UNIFESP-EPM
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Regular Research Grants
• FAPESP's process: 05/03514-9 - Studies of the physiological function and biotechnological potential of protease inhibitors and anti-hemostatics in hematophagous arthropods
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Research Projects - Thematic Grants