Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity

Abstract

The positive charge concentrated at the C-terminal region of ammodytoxin (Atx) A, which is involved in presynaptic toxicity, has been reversed. A six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E, where K108N = Lys108 → Asn etc.) was prepared, in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones. The mutant was approximately 30-fold less lethal, but still neurotoxic. Consistent with this, its binding affinity for the neuronal receptors decreased by only a factor of five. Additionally, a single-site mutant of AtxA was prepared, with substitution at only one position (K127T) out of six mutated in the six-site mutant. Its toxicity indicated that most, if not all, of the six mutated residues partially contribute to the decreased lethality of the multiple-site mutant.

Ammodytoxins, Potent Presynaptic Neurotoxins, Are Also Highly Efficient Phospholipase A2 Enzymes

“…AtnI 2 is a neutral protein (pI 6.8, net charge 0) (55) that contains Trp at position 31, and the overall nature of its IBS is more hydrophobic than that of AtxA (Figures 1 and 5). Therefore, the high activity of AtnI 2 on zwitterionic POPC vesicles was expected and is in accordance with its high activity on mixed micelles of egg yolk PC and Triton X-100 (55). AtnI 2 also showed high activity on anionic POPG vesicles but surprisingly low affinity and activity on PS-containing vesicles and cell membranes.…”