Tyr-->Trp-substituted peptide 115-129 of a Lys49 phospholipase A(2) expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect.
Myotoxin II is a group II Lys49 phospholipase A(2) (PLA(2)) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115-129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA(2)-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115-129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism.
Lomonte
B
B
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. blomonte@cariari.ucr.ac.cr
Pizarro-Cerdá
J
J
Angulo
Y
Y
Gorvel
J P
JP
Moreno
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000097:blood; D003435:Crotalid Venoms / Q000737:chemistry; D004727:Endothelium / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects; D054501:Group II Phospholipases A2; D007124:Immunoenzyme Techniques; D007273:Injections, Intramuscular; D008239:Lysine / Q000737:chemistry; D008566:Membranes / Q000187:drug effects; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000473:pathology; D009498:Neurotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D030162:Reptilian Proteins; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D003435:Crotalid Venoms; D009498:Neurotoxins; D010446:Peptide Fragments; D030162:Reptilian Proteins; D014443:Tyrosine; D014364:Tryptophan; D003402:Creatine Kinase; D010741:Phospholipases A; D054501:Group II Phospholipases A2; C061056:myotoxin II, Bothrops asper; D008239:Lysine