Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A.
The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-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 beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.
Ivanovski
Gabriela
G
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Petan
Toni
T
Krizaj
Igor
I
Gelb
Michael H
MH
Gubensek
Franc
F
Pungercar
Joze
J
D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000737:chemistry*; D000818:Animals; D001923:Brain Chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics; D010740:Phospholipases / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D017729:Presynaptic Terminals / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D016415:Sequence Alignment; D013552:Swine; D014757:Viper Venoms / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*
D024361:Amino Acids, Basic; D009498:Neurotoxins; D011994:Recombinant Proteins; D014757:Viper Venoms; C068117:ammodytoxin A; D010740:Phospholipases