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Crotoxin and taipoxin are both neurotoxic phospholipases A 2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A 2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A 2 . Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A 2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca 2+ -free condition, taipoxin or crotoxin inhibits with ic 50 of 20–1000 nM the Na + -dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected. Furthermore, the high-affinity site for [ 3 H]desipramine binding, known to be the norepinephrine transporter, is inhibited with an ic 50 of 14nM by taipoxin independent of phospholipase activity. These results are strong indications that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modification at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Phe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an ic 50 of 1 μM for the binding of [ 125 I]crotoxin, contrasting sharply with the complete lack of such ability with the wild type even at 50 μM. In addition, rat phospholipase A 2 , which has a corresponding Tyr residue, can inhibit the binding of [ 125 I]crotoxin.

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A2 variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng, M.-C.(1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A2 generated by site-directed mutagenesis of Phe of the wild-type enzyme to Tyr. The IC of this inhibition was estimated to be 1 μM. In sharp contrast, the wild-type enzyme gave no effect even at 50 μM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A2 molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A2 variants to the 45-kDa receptor.

The subtelomere and a portion of the associated telomeric region (together named 3RTAS) of chromosome IIIR from the Arabidopsis thaliana ecotypes Columbia (Col) and Wassilewskija (Ws) were specifically amplified by polymerase chain reaction and subsequently cloned and sequenced. The centromere-proximal portion of 3RTAS from both ecotypes contained two newly identified potential genes, one encoding the chloroplast luminal 19-kDa protein precursor and the other encoding three potential alternatively spliced CCCH-type zinc finger proteins. The telomere-proximal portion of 3RTAS from the Col ecotype contained short duplicated fragments derived from chromosomes I, II, and III, and that from the Ws ecotype contained a duplicated fragment derived from chromosome V. Each duplicated fragment has diverged somewhat in sequence from that of the ectopic template. Small patches of homologous nucleotides were found within the flanking sequences of both the duplicated fragments and the corresponding ectopic template sequences. The structural characteristics of these duplicated fragments suggest that they are filler DNAs captured by non-homologous end joining during double-strand break repair. Our characterization of 3RTAS not only filled up a gap in the chromosome IIIR sequence of A. thaliana but also identified new genes with unknown functions.

Trimucrotoxin (TmCT) is an Asn(6)-containing phospholipase A(2) (PLA(2)) from Protobothrops mucrosquamatus (pit-viper) venom. In an attempt to characterize the amino acid residues responsible for the neurotoxic and anticoagulant activities of TmCT, the recombinant fusion proteins of TmCT wild type and mutants were expressed in Escherichia coli. Correct refolding and processing of 37 TmCT mutants were confirmed by their HPLC retention times, circular dichroism spectra, and masses obtained from ESI-MS spectrometry. Each mutant was assayed by pH-stat titration using zwitterionic as well as anionic micelle substrates, and the neurotoxicity was evaluated by using the contractile responses of chick biventer cervicis muscles. The results demonstrated that the residues Asn(1), Asn(6), Lys(7), Ile(11), Met(12), Gly(53), Thr(79), His(108) and Met(118) are important to TmCT neurotoxicity. Through various tests, we also confirmed that enzymatic activity, as opposed to binding to Factor Xa, was a necessary part of TmCT's anticoagulant effect. In addition, pulldown assays of the WT and selected mutants revealed that TmCT's in vitro binding to crotoxin acidic subunit may involve a broad surface area. We conclude that the hot spot mutations at specific positions 53, 79, 108, and 118 during the pit-viper Asn(6)-PLA(2) evolution regulate their neurotoxicities, and that many of the neurotoxic site residues and the anticoagulant mechanism of TmCT are different from those of ammodytoxin A (a true-viper venom neurotoxic PLA(2)).

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Some myotoxic or neurotoxic PLA2s (phospholipases A2) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA2s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA2s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA2s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA2s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA2s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA2s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA2s. Structure–activity relationships at various regions of the PLA2 molecules were extensively discussed.

Binding proteins in neuronal membranes for a phospholipase A2 with presynaptic neurotoxicity have been purified. Three polypeptides of 87, 65, and 50 K Da were obtained from the synaptic membrane fraction of guinea pig brain utilizing an immobilized crotoxin (a phospholipase A2) column. For large scale purification, porcine brain was used instead, and two polypeptides of 50 and 18 K Da were found. The 65 and 18 K polypeptides may represent hitherto unidentified components of the crotoxin-binding proteins. Partial N-terminal amino acid sequence and a partial sequence for an internal peptide fragment have been determined for the 50 K polypeptide. Search of protein data bank reveals that this polypeptide or protein is a novel member of the reticulocalbin family of calcium-binding proteins.

Snake presynaptic toxins such as crotoxin, β-bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A2 activities. On the other hand, many other phospholipase A2s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, β-bungarotoxin and taipoxin were first radioactively labeled with Na(125I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A2. Crotoxin and taipoxin, but not β-bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (125I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.

Summary Affinity labeling techniques were used to identify the neuronal membrane molecules involved in the binding of taipoxin, a neurotoxic protein with phospholipase A 2 activity. After [ 125 I]taipoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate resulted in the formation of a predominant radioactive conjugate of 60,000 Da. Notexin and some other PLA 2 s are weakly inhibitory to this conjugation, while β-bungarotoxin and some others are not inhibitory. The 60K conjugate was not detected when plasma membranes from several nonneuronal tissues were used. We concluded that a 45,000 Da protein specifically present in neuronal membranes is (a subunit of) the major molecule responsible for taipoxin binding.

Utilizing Marathon-ready cDNA library and a gene-specific primer corresponding to a partial amino acid sequence determined previously, the complete nucleotide sequence for the cDNA of crocalbin, which binds crotoxin (a phospholipase A2) and Ca2+, was obtained by polymerase chain reaction. The open reading frame of the cDNA encodes a novel polypeptide of 315 amino acid residues, including a signal sequence of 19 residues. This protein contains six potential Ca2+-binding domains, one N-glycosylation site, and a large amount of acidic amino acid residues. The ability to bind Ca2+ has been ascertained by calcium overlay experiment. Evidenced by sequence similarity in addition, it is concluded that crocalbin is a new member of the reticulocalbin family of calcium-binding proteins.