Steven D. Aird

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.

Prey specificity, comparative lethality and compositional differences of coral snake venoms

Toxicities of crude venoms from 49 coral snake (Micrurus sp.) populations, representing 15 nominal taxa, were examined in both laboratory mice and in native prey animals and compared with data gathered from two non-micrurine elapids and a crotalid, which served as outgroups. These venoms were further compared on the basis of 23 enzymatic activities. Both toxicities and enzymatic activities were analyzed with respect to natural prey preferences, as determined from stomach content analyses and literature reports. Venoms of nearly all Micrurus for which prey preferences are known, are more toxic to natural prey than to non-prey species. Except for amphisbaenians, prey are more susceptible to venoms of Micrurus that feed upon them, than to venoms of those that eat other organisms. All venoms were more toxic i.v.>i.p.>i.m. Route-specific differences in toxicity are generally greatest for preferred prey species. Cluster analyses of venom enzymatic activities resulted in five clusters, with the fish-eating M. surinamensis more distant from other Micrurus than even the crotalid, Bothrops moojeni. Ophiophagous and amphisbaenian-eating Micrurus formed two close subclusters, one allied to the outgroup species Naja naja and the other to the fossorial, ophiophagous Bungarus multicinctus. Prey preference is shown to be the most important determinant of venom composition in Micrurus.

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.

Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis)

BackgroundAdvances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology.ResultsSequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals.ConclusionsThis study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species.

Comparative enzymatic composition of Brazilian coral snake (Micrurus) venoms

1. Venoms of 11 coral snake taxa, including Micrurus albicinctus, M. corallinus, M. frontalis altirostris, M. f. brasiliensis, M. f. frontalis, M. fulvius fulvius, M. ibiboboca, M. lemniscatus ssp., M. randonianus, M. spixii spixii, and M. surinamensis surinamensis, were examined for 13 enzymatic activities. 2. These were compared with venoms of three outgroup taxa: Naja naja kaouthia, Bungarus multicinctus, and Bothrops moojeni. 3. Enzyme activity levels in Micrurus venoms were highly variable from species to species. 4. All venoms possessed phospholipase activity. 5. Protease activity against synthetic or dyed natural substrates was generally negligible in all elapid venoms examined. By contrast, most Micrurus venoms displayed ample L-leucine aminopeptidase activity. 6. Venom of M.s. surinamensis was significantly different from those of its congeners in most assays.

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.

Primary structure of γ-bungarotoxin, a new postsynaptic neurotoxin from venom of Bungarus multicinctus

The primary structure of gamma-bungarotoxin, a new toxin from Bungarus multicinctus venom, was determined using mass spectrometry and Edman degradation. The toxin has a mass of 7524.7 D and consists of 68 residues having the following sequence: MQCKTCSFYT CPNSETCPDG KNICVKRSWT AVRGDGPKRE IRRECAATCP PSKLGLTVFC CTTDNCNH. Gamma-bungarotoxin is structurally similar to both kappa-bungarotoxin and elapid long postsynaptic neurotoxins. Its C-terminal nine residues are identical to those of the kappa-toxins. Its disulfide bond locations appear identical to those of several elapid toxins of unknown pharmacology and its hydrophobicity profile is also strikingly similar. However, with an LD50 of 0.15 microg/g i.v. in mice, gamma-bungarotoxin is 30-150-fold more toxic than other members of this latter class. Its toxicity is comparable to those of alpha-nicotinic acetylcholine receptor antagonists.

A crotoxin homolog from the venom of the Uracoan rattlesnake (Crotalus vegrandis).

A major protein toxin from the venom of Crotalus vegrandis was examined by gel filtration, anion-exchange chromatography, and SDS polyacrylamide gel electrophoresis. The toxin was separated into several isoforms by ion-exchange chromatography and spontaneously dissociated into free acidic and basic subunits, mimicking the behavior of crotoxin. Rabbit antisera raised against crotoxin reacted strongly in enzyme-linked immunosorbent assays with the intact C. vegrandis toxin isoforms and their basic subunits, and formed precipitin lines of identity with intact crotoxin in double immunodiffusion gels. These results indicate that vegrandis toxin is strongly homologous with crotoxin from the venom of Crotalus durissus terrificus.