Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes.
A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). The putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. In this work, we reported the biochemical characterization of BmCI. The recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of 1mgL(-1) of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K(i) (6.2pM). The BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. The cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC(50)=8microM). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. In conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity.
Lima
Cássia A
CA
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, São Paulo, SP, Brazil.
Torquato
Ricardo J S
RJ
Sasaki
Sergio D
SD
Justo
Giselle Z
GZ
Tanaka
Aparecida S
AS
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D007611:Aprotinin / Q000737:chemistry / Q000378:metabolism*; D051940:B7-2 Antigen; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation; D006434:Hemocytes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D048494:Rhipicephalus / Q000378:metabolism* / Q000382:microbiology
D051940:B7-2 Antigen; C497964:CD86 protein, human; D007611:Aprotinin