THE C-TERMINAL LOOP OF BOTHROPSTOXIN-I, A LYS-49 PLA2 FROM Bothrops jararacussu, IS A STRUCTURAL DETERMINANT OF PHAGOCYTOSIS STIMULATION IN MACROPHAGES.

Zuliani, J.P.¹, Chioato, L²., Fernandes, C.M.¹, Ward, R.J³.; Teixeira, C.F.P¹.

1 Lab. Farmacologia – Instituto Butantan, 2 Dep. Bioquimica e Imunologia, FMRP-USP, 3 Dep. Quimica, FFCLRP-USP.

Bothropstoxin-I (BthTx-I) displays myotoxic and in vitro membrane-damaging activities. The C-terminal region of this PLA2 is essential for these activities. In this study we have evaluated the effect of native and recombinant BthTx-I, the active site mutant H48Q and the C-terminal mutants K115A, K116A, Y117W, R118A, Y119W, K122A F125W and K129A on mannose receptor-mediated phagocytosis in macrophages. Macrophages were obtained from Swiss mice peritoneum 96 hours after i.p. injection of 3% thioglycollate. Cell viability after toxin treatment was measured by Trypan blue exclusion test. Phagocytosis was studied with non-opsonized zymosan. C-terminal mutants were obtained by site-direct mutagenesis of recombinant BthTx-I (rBthTx-I) expressed in E. coli, and purified by cationic exchange chromatography. All recombinant proteins demonstrated native-like secondary structure as evaluated by circular dichroism spectroscopy. Results showed that although the rBthTx-I, H48Q and Y117W mutants demonstrated a slight decrease in cell viability (15-18%) in macrophages, the native BthTx-I, rBthTx-I, H48Q, K115A, Y117W, Y119W, K122A and K129A but not K116A, R118A and F125W mutants significantly increased phagocytosis. These findings indicate that BthTx-I and mutants are at most only weakly cytotoxic for macrophages, and that specific C-terminal mutants abolish the stimulation of phagocytosis, demonstrating that cationic and aromatic amino acids in the C-terminal region contribute as structural determinants of this effect in BthTx-I.

Financial Support: FAPESP (02/01009-7; 01/00279-8), CNPq, PRP-USP.

Correspondence to: jzuliani@butantan.gov.br