Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A(2): Role of negatively charged phospholipids as potential membrane binding sites

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp- 49 and Lys-49 phosphollipases A(2) (PLA2s), which drastically differ in enz ymatic activity, were devoid of direct hemolytic effects on erythrocytes (R BC) from different species despite the fact that enzymatically active CGMT- I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes pr epared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent, Unlike normal RBC, si gnificant hemolysis was induced by CGMT-II and another similar Lys-49 isofo rm, B, asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS), Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PD A), a potent inhibitor of aminophospholipid transport. RBC enriched with ph osphatidic acid (PA);,iso became susceptible to the myotoxins but was unaff ected by PDA, Cells enriched with phosphatidylcholine (PC) remained resista nt to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone, When RBC binding of BAMT-II wa s measured by enzyme-linked immunosorbent assay, it was observed that PS- a nd PA-enriched erythrocytes were always able to capture more toxin than nor mal and PC-enriched RBC, This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absenc e of calcium in the medium. These data suggest that negatively charged lipi ds in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC co uld explain their resistance to the action of these PLA2s, (C) 2001 Academi c Press.