Ivan I. Kaiser

Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2.

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.

The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper

A myotoxic, basic phospholipase A2 (pI greater than 9.5) with anticoagulant activity has been purified from the venom of Bothrops asper, and its amino acid sequence determined by automated Edman degradation. It is distinct from the B. asper phospholipase A2 known as myotoxin I [Lomonte, B. and Gutierrez, J. M., 1989, Toxicon 27, 725] but cross-reacts with myotoxin I rabbit antisera, suggesting that the proteins are closely related isoforms. To our knowledge, this is the first myotoxic phospholipase to be sequenced that lacks presynaptic neurotoxicity (iv LD50 approximately equal to 8 micrograms/g in mice). The protein appears to exist as a monomer, contains 122 amino acids, and fits with subgroup IIA of other sequenced phospholipase A2 molecules. Its primary sequence shows greatest identity with ammodytoxin B (67%), a phospholipase A2 presynaptic neurotoxin from Vipera ammodytes ammodytes venom. Hydropathy profiles of B. asper phospholipase and the ammodytoxins also show great similarities. In contrast, even though the amino acid sequence identities between B. asper phospholipase and the basic subunit of crotoxin remain high (64%), their hydropathy profiles differ substantially. Domains and residues that may be responsible for neurotoxicity are discussed.

Comparative studies on three rattlesnake toxins

Toxins from the venoms of Crotalus durissus terrificus, Crotalus s. scutulatus and Crotalus viridis concolor were compared using gel filtration, ion-exchange chromatography on DEAE-Sephacel and denaturing and non-denaturing polyacrylamide gel electrophoresis. The three heterodimeric native toxins behaved similarly on each of the separation media, except that the C. d. terrificus toxin displayed a pronounced tendency to dissociate on DEAE-Sephacel, even in the absence of urea. In the presence of 6M urea, subunit dissociation was quantitative for all three toxins. Recombination of purified subunits resulted in toxins which eluted from the gel filtration column in identical fashion to native toxins. Non-denaturing polyacrylamide gel electrophoretic patterns of recombined toxins actually showed greater band resolution than did the native toxins. Six hybrid toxins were generated on polyacrylamide gels from cross-combinations of purified subunits, each with different mobilities than the parental toxins. Mobilities of the hybrid toxins depended principally upon the mobilities of the basic subunits. All three purified native toxins showed comparable LD50s in female mice (0.039-0.061 micrograms/g). The C. d. terrificus acidic X C. s. scutulatus basic hybrid toxin showed toxicity identical to that of the C. s. scutulatus recombined toxin. Phospholipase activity is associated with the basic subunit in all three toxins. Intact toxins show a distinctive lag in phospholipase activity which is not seen with purified basic subunits alone. These results indicate that the principal toxins in these three venoms are homologous.

A complete amino acid sequence for the basic subunit of crotoxin

The complete amino acid sequence of the basic subunit of crotoxin from the venom of Crotalus durissus terrificus has been determined. Fragmentation of the protein was achieved by using cyanogen bromide and arginine- and lysine-specific endoproteases. Sixteen Glx and Asx residues reported by Fraenkel-Conrat et al. (1980) in Natural Toxins (D. Eaker and T. Wadstrom, eds.), pp. 561-567, Pergamon, Oxford.) have been resolved as Glu or Gln and Asp or Asn residues, respectively. Most of the remaining sequence is identical to that reported by the foregoing authors although several significant differences were evident in our protein. Tyr-61 was not present; thus the correct sequence is Lys-60, Trp-61. The latter sequence aligns with sequences of all other known viperid and crotalid phospholipases A2 (S. D. Aird, I. I. Kaiser, R. V. Lewis, and W. G. Kruggel (1985) Biochemistry 24, 7054-7058). Other differences include Asx-99, which is Ser, and Asx-105, which is Tyr. Some positions display allelic variation. In some lots of venom Glx-33 is Gln, while in others it is Arg. Positions 37 and 69 occur as mixtures of both Lys and Arg. Amino acid sequence comparisons between the basic and acidic subunits of crotoxin and between the basic subunit and other phospholipase A2 molecules indicate that the basic subunit is structurally most similar to the monomers of nontoxic, dimeric phospholipases A2 from the venoms of Crotalus adamanteus, Crotalus atrox, and Trimeresurus okinavensis, and to the toxic monomeric phospholipase A2 from the venom of Bitis caudalis.

Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Influence of ionizing radiation on crotoxin: Biochemical and immunological aspects

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35, 1193-1203, 1997.-Two sets of proteins have been purified from the venom of the Brazilian coral snake, Micrurus frontalis frontalis. One set has mol. wts, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the 8000-13,000 range and includes some proteins which are toxic to mice and others which are not. These proteins appear to be isoforms of postsynaptic toxins. The other set shows phospholipase A2 (PLA2) activity and the toxic members of this set promote hemorrhage in mice in a manner closely resembling that produced by PLA2s isolated from the venom of the Australian tiger snake (Notechis scutatus scutatus). These PLA2s migrate on SDS-PAGE with apparent mol. wts in the 18,000-22,000 range which is characteristic of PLA2s that have an alpha-helix D similar to pancreatic PLA2s. Elapid venom PLA2s of the type which typically migrate on SDS-PAGE with mol. wts in the 13,000-16,000 range and do not have alpha-helix D have not been detected in M. f. frontalis venom.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

A GYROXIN ANALOG FROM THE VENOM OF THE BUSHMASTER (LACHESIS MUTA MUTA)

Clinical observations of possible neurotoxic activity in bushmaster (Lachesis muta muta) envenomations, coupled with the accepted ancestral relationship of Lachesis to other crotalids, suggested that Lachesis venom might contain a crotoxin-like molecule. Crude venom and gel-filtration fractions showed modest reactivity in enzyme-linked immunosorbent assays using rabbit polyclonal antibodies raised against the basic subunit of crotoxin, but no reaction was detected with a murine monoclonal antibody raised against the same antigen. Phospholipase assays, LD50 determinations and SDS-polyacrylamide gel electrophoresis indicated the presence of non-toxic phospholipases, but no crotoxin homologs. A higher mol.wt, toxic protein (60,000) with an LD50 of 0.07 micrograms/g in mice was isolated and purified, which induced gyroxin-like, rapid rolling motions in mice. Its amino terminal sequence shows considerable amino acid sequence identity with gyroxin from the venom of Crotalus durissus terrificus and other serine proteases.

THE AMINO ACID SEQUENCE OF THE ACIDIC SUBUNIT B-CHAIN OF CROTOXIN

The B-chain of the acidic subunit of crotoxin proved refractory to Edman degradation. When subjected to sequence analysis using tandem mass spectrometry, pyroglutamate was found at the amino-terminal end, even though earlier attempts to de-block with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity with the corresponding segment from Mojave toxin, a homologous neurotoxin from Crotalus scutulatus scutulatus. The sequence of the last 24 residues of the B-chain is consistent with that previously published (Aird, S.D., Kaiser, I.I., Lewis, R.V. and Kruggel, W.G. (1985) Biochemistry 24, 7054-7058), except at position 20, where Edman degradation gave glycine and mass spectrometry gave glutamic acid.