title: Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut.
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 37.5mg/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.
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
authors.forename: Tatiane Sanches
authors.initials: TS
authors.lastname: Soares
authors.forename: Renata Midori Okuta
authors.initials: RM
authors.lastname: Watanabe
authors.forename: Anita Mitico
authors.initials: AM
authors.lastname: Tanaka-Azevedo
authors.forename: Ricardo José Soares
authors.initials: RJ
authors.lastname: Torquato
authors.forename: Stephen
authors.initials: S
authors.lastname: Lu
authors.forename: Ana Carvalho
authors.initials: AC
authors.lastname: Figueiredo
authors.forename: Pedro José Barbosa
authors.initials: PJ
authors.lastname: Pereira
authors.forename: Aparecida S
authors.initials: AS
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D041981:Gastrointestinal Tract / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D008969:Molecular Sequence Data; D034622:RNA Interference; D048494:Rhipicephalus / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D000941:Antigens; D060829:Arthropod Proteins; D013917:Thrombin