Basic amino acid residues in the β-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

• Published in: Biochimica et biophysica acta Vol. 1702; no. 2; pp. 217 - 225
• Main Authors: Ivanovski, Gabriela, Petan, Toni, Križaj, Igor, Gelb, Michael H., Gubenšek, Franc, Pungerčar, Jože
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-11-2004
• Subjects: Amino Acid Sequence; Amino Acids, Basic - chemistry; Animals; Brain Chemistry; Enzymatic activity; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurotoxicity; Neurotoxins - chemistry; Neurotoxins - genetics; Phospholipase A 2; Phospholipases - chemistry; Phospholipases - genetics; Phospholipases - toxicity; Presynaptic Terminals - drug effects; Protein Structure, Tertiary; Receptor-binding affinity; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - toxicity; Sequence Alignment; Site-directed mutagenesis; Swine; Viper Venoms - chemistry; Viper Venoms - genetics; Viper Venoms - toxicity; Vipera ammodytes ammodytes; Phospholipase A 2; Atx; Vipera ammodytes ammodytes; POPC/G; FABP; R25 and R180; Receptor-binding affinity; sPLA 2; LD 50; CaM; Neurotoxicity; Enzymatic activity; Site-directed mutagenesis; IBS; PLA 2
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2004.09.002

The molecular mechanism of action of presynaptically toxic secreted phospholipases A
2 (sPLA
2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the β-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the β-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.