Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus)

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.

The non-phospholipase A2 subunit of β-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit

Three isotoxins (SP I-III) of the beta-bungarotoxin family were purified to homogeneity via a series of isolation procedures including a final step of h.p.l.c. on an SP column washed with a linear gradient of 0.2-0.6 M sodium acetate at pH 7.4. Their proportions varied greatly with the batch of venom. Each isotoxin was demonstrated by SDS/PAGE to contain a phospholipase A2 subunit and a non-phospholipase A2 subunit. The three proteins were reductively alkylated with 4-vinylpyridine and the alkylated derivatives of the two subunits of each isotoxin were separated. N-Terminal sequence analysis of the alkylated derivatives revealed that the three isotoxins probably share a common phospholipase A2 subunit but differ in their non-phospholipase A2 subunits. The non-phospholipase A2 subunits of SP II and SP III were identical with those of beta 2- and beta 1-toxin respectively, except that there was an additional valine inserted between Thr-18 and Val-19 in beta 2-toxin and Pro-18 and Val-19 in beta 1-toxin. The non-phospholipase A2 subunit of SP I differed greatly from that of SP III but was almost identical with that of SP II, except that Lys-14 and Ala-29 in SP II were replaced by Arg-14 and Glu-29 in SP I. Analysis of the effect of CaCl2 on protein fluorescence showed the existence of a low- and a high-affinity site on the different domains of each isotoxin for Ca2+ binding. The three isotoxins showed no great difference in their ability to bind Ca2+ on both the high- and low-affinity site. They had slightly different phospholipase A2 activities but differed to a great extent with respect to their neurotoxic effects. LD50 values increased in the order SP I > SP II > SP III. In contrast, the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle was in the order SP III > SP II > SP I.

Met-8 of the β1-bungarotoxin phospholipase A2 subunit is essential for the phospholipase A2-independent neurotoxic effect

beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. The toxin was oxidized with a 100-fold molar excess of chloramine T with respect to the methionine content of the protein in 0.1 M Tris/HCl at pH 8.5 and at room temperature. Reactivities of the two methionine (Met-6 and Met-8 of the phospholipase A2 subunit), five histidine, 14 tyrosine and one tryptophan residues of one toxin molecule with chloramine T were assessed from the change in intrinsic fluorescence and amino acid composition of the protein. Met-8 and one tyrosine on the phospholipase A2 subunit and less than one histidine were oxidized, while Met-6 remained intact after 30 min of reaction. One histidine and approx. two tyrosine residues were oxidized when both methionine residues were oxidized after 90 min of reaction. The sole tryptophan was oxidized slightly throughout the reaction. The chloramine T oxidation did not destroy the two Ca(2+)-binding domains, though it modified the toxin to become less effective at binding Ca2+. The modified toxin obtained after 30 or 90 min reaction time retained 65% or 40% of the phospholipase A2 activity of the parent toxin, but both were not lethal to mice and showed a very weak ability to induce the indirectly evoked contraction of chick biventer cervicis muscle. It is suggested that Met-8 may play an important role in the phospholipase A2-independent interaction with the nerve terminal membrane during the neurotoxic effect of beta 1-bungarotoxin.