2 DISTINCT TYPES OF CARDIOTOXIN AS REVEALED BY THE STRUCTURE AND ACTIVITY RELATIONSHIP OF THEIR INTERACTION WITH ZWITTERIONIC PHOSPHOLIPID DISPERSIONS

Cardiotoxins (CTXs) are a group of homologous proteins found in cobra snake venom and consist of 60-62 amino acid residues. Although CTXs ar e known to consist of three extended beta-sheet loops similar to neuro toxins, the target and interaction of CTXs with membranes unlike those of neurotoxins are not well understood. Herein, we report comparative studies of 10 CTXs purified from Taiwan cobra (Naja naja atra) and Mo zambique spitting cobra (Naja mossambica mossambica) snake venoms with respect to their interactions with zwitterionic phospholipids. Based on the CTX-induced mixing of sphingomyelin vesicles and the binding of CTX to lysophosphatidylcholine micelles, two distinct types of CTX, i .e. P- and S-type CTX, are identified. P-type CTXs are characterized b y the presence of Pro-31 within a putative phospholipid binding site n ear the tip of loop 2; whereas S-type CTXs are characterized by the pr esence of Ser-29 within the same but more hydrophilic region. Although binding of all CTXs to phospholipid membranes involves a phospholipid binding site at loop 1, P-type CTXs exhibit higher fusion and binding activity than S-type CTXs, presumably due to the additional phospholi pid binding site at loop 2. The binding modes of P- and S-type CTX are thus different. Analysis of the primary structures of 46 CTXs from th e genus Naja indicates that these two types of CTXs exist in all speci es examined. Reasonable structure/activity correlation can be detected for the effects of CTXs on muscle and red blood cells, although notab le exceptions are also found. S-type CTXs are generally found to exhib it higher muscle cell depolarization activity, whereas P-type CTXs are found to possess a higher hemolytic activity. Thus the mechanism of a ction of CTXs seems to involve CTX-membrane interactions and depends o n the type of the cell membrane and CTX molecules under study. The two lipid binding sites in P-type CTXs and one lipid binding site in S-ty pe CTXs show large variation in their amino acid residues, but they do display some common distribution of residue type. Analogous to the si gnal sequences for protein import, these regions are characterized by the coexistence of an exposed hydrophobic surface flanked on either si de by a cationic residue. A hypothesis is proposed to explain the gene ral cytotoxic and specific cardiotoxic effect of CTXs based on the two CTX subtypes in snake venom.