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 phospholipases A(2) (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared 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, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant 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 was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal 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 absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.
Díaz
C
C
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. cdiaz@cariari.ucr.ac.cr
León
G
G
Rucavado
A
A
Rojas
N
N
Schroit
A J
AJ
Gutiérrez
J M
JM
D000818:Animals; D017837:Bothrops; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D054501:Group II Phospholipases A2; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008560:Membrane Fluidity / Q000187:drug effects; D009439:Neuraminidase / Q000378:metabolism; D009498:Neurotoxins / Q000378:metabolism / Q000633:toxicity; D010741:Phospholipases A / Q000378:metabolism / Q000633:toxicity*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D030162:Reptilian Proteins; D013045:Species Specificity
D003435:Crotalid Venoms; D008051:Lipid Bilayers; D008081:Liposomes; D009498:Neurotoxins; D010743:Phospholipids; D030162:Reptilian Proteins; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C061056:myotoxin II, Bothrops asper; C113262:myotoxin II, Bothrops godmani; D009439:Neuraminidase