An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins

Abstract

Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124-->I/K128-->E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128-->E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108-->N/K111-->N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128-->E) is equivalent to the AtxC(I124-->F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.

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

“…The most toxic AtxA is 17 times more toxic than AtxC, a natural AtxA(F124I\K128E) mutant, and 28 times more toxic than AtxB, a natural AtxA(Y115H\R118M\N119Y) mutant [10]. This was later confirmed by site-specific antibodies directed toward the C-terminal-region epitopes, which neutralized the lethality of Atxs [11,12], and by site-directed mutagenesis [13]. Most presynaptically neurotoxic PLA # s are highly basic proteins (pI 9).…”

“…AtxA and the AtxA(K108N\K111N) mutant were produced in Escherichia coli and purified as described in [13]. Restriction enzymes, Taq polymerase and T7 DNA ligase were obtained from Boehringer Mannheim and Pharmacia.…”

“…Mutagenesis was performed by PCR using the expression plasmid encoding either wild-type AtxA or the AtxA(K108N\K111N) mutant as a template [13]. The amplification mixture (100 µl) consisted of 50 mM KCl, 10 mM Tris\HCl (pH 8.3), 1.5 mM MgCl # , 200 µM each of four deoxynucleoside triphosphates, 50 ng of the plasmid, 400 nM each primer and 2.5 units of Taq polymerase.…”

“…After PCR (67 mC for 45 s, 72 mC for 60 s and 95 mC for 45 s for 30 cycles), the PCR products of 390 bp were purified by Geneclean II (BIO101). Following digestion with PstI and HindIII, the restriction fragments coding for the C-terminal half of AtxA with the generated mutations were respectively inserted into the PstIHindIII opened expression plasmid, which still contained the coding sequence for the N-terminal half of AtxA [13]. The sequences of the two constructs were confirmed by dideoxynucleotide sequencing [16].…”

“…6.5 g of wet bacterial cells\l of fermentation broth was obtained. Inclusion bodies were isolated and the proteins were refolded and activated as described previously [13]. The mixtures containing activated proteins were dialysed against 20 mM sodium acetate, pH 4.8, and the mutants purified by FPLC on a Mono S column (HR 5\5 ; Pharmacia).…”