BIOLOGICALLY ACTIVE AGENTS IN NATURE 56 CHIMIA 52 (1998) NT. 1/2 (lnnu'T/FehruaT) Chimia 52 (1998) 56-62 and also on the postsynaptic membrane © Neue Schweizerische Chemische Gesellschaft [7], in addition to the presynaptic binding ISSN 0009-4293 site. From a structural viewpoint, both subunits A and B are in the isoforms [8a- c]. Neurotoxins from Snake Venom Mojave toxin from Crotalus scututa- tus is also structurally similar to crotoxin Anthony T. Tu* and is composed of two subunits [9a, b]. There are considerable amino-acid se- quence homologies between the two tox- Abstract. Found within snake venoms are a variety oftoxic and nontoxic proteins. The ins [lOa, b]. Mojave toxin inhibits calci- effects of snake venoms depend on all of the components of that venom. However, the um-channel dihydropyridine receptor bind- lethal effects are usually related to the most potent toxins found within the venom; the ing in rat brain [1 I]. two most toxic proteins found in snake venom are neuro- and cardiotoxins. This article Crotoxin's neurotoxic action is very reviews a variety of neurotoxins found in snake venom. The range of neurotoxins similar to t3-btx, but has some difference. present in snake venom is varied and includes: peripheral and noncentral, presynaptic, For instance, crotoxin and its subunit B postsynaptic, potassium-channel inhibiting, muscarinic transmission inhibiting, and have a postsynaptic effect, whereas t3-btx anticholinesterase types. has no such activity [7]. The acidic and basic subunit-types pre- Because a snake venom contains many turn causes the exocytosis of the nerve synaptic toxins are fairly common in neu- different proteins, some are highly toxic, transmitter. The exocytosis is made by the rotoxic snake venoms. For instance, such some are weakly toxic, and some are non- membrane fusion of the vesicle and the toxins have been isolated from the venoms toxic. Toxicity of a snake venom is due to nerve-terminal membranes. In vitro, it was of C. viridis conca/or [12] and C. durissus a combinational effect of all components demonstrated that t3-btx mediated the fu- collilineatus [13]. The amino-acid se- present. However, some snake venoms are sion of liposome. The result of in vitro quence of the basic subunit also has con- more toxic than others because they con- fusion may explain in vivo exocytosis [I]. siderable homology to other snake-venom tain more lethal components. In this re- This can be clearly seen by observing phospholipases A. view article, the most toxic neurotoxin is the change in the miniature end-plate po- The second type of presynaptic neuro- discussed. tential (MEPP). The MEPP is a very small toxin from a chemical viewpoint is that There are several types of neurotoxins potential, observed in the neuromuscular two polypeptide chains are connected by a and their structures; the site of action and junction, that is due to the natural leakage disulfide bond. The most typical toxin of the mechanism are not identical. Snake of acetylcholine from the vesicle. When t3- this type is f3-btx. It consists of two chains: neurotoxins are peripheral neurotoxins, toxin is applied, the MEPP frequently de- The A-chain has 120 amino acids, and the rather than centrally neurotoxic; apparent- creases first (5-10 min), then suddenly B-chain has 60 amino-acid residues. The ly, they do not pass through the blood- increases (for several hours). Finally, the amino-acid sequence of the A-chain is brain barrier. frequency decreases until it becomes zero. similar to the phospholipase A sequence There are several types of presynaptic and, in fact, the A-chain does possess toxins. They are usually structurally dis- phospholipase A activity. For presynaptic 1. Presynaptic Neurotoxins tinct among themselves. However, there activity, phospholipase A activity is es- is one common property, and that is the sential. For instance, when Ca2+ is re- The presynaptic neurotoxins are also possession of phospholipase A activity. placed with Sr2+, phospholi pase A acti vity called t3-toxins in contrast to postsynaptic The toxic phospholipase A is usually a and presynaptic activity are both lost. or a-toxins. When a t3-toxin is added to the basic protein. Tsai et at. [2] found that the Oxidation of methionine at the 6- and neuromuscular preparation, the muscle basic amino acid tended to cluster near the 8-positions lowered the toxicity without contraction starts without stimulation of surface region at the NHTterminal side in affecting antigenicity. Moreover, theNH2- the nerve axon. t3-Toxin usually does not basic phospholipase A. terminal region of the A-chain plays a affect the depolarization of the muscle One type of presynaptic toxin is com- crucial role in maintaining functional ac- itself or have a binding ability to the ace- posed oftwo subunits bound together. The tivity [14]. Heterodimeric f3-btx was ex- tylcholine receptor. It is thus clear that the basic subunit has phospholipase A activi- amined by X-ray diffraction [15]. f3-toxin somehow affects the presynaptic ty, whereas the acidic subunit has no en- The exact mechanism of t3-btx is not end of the nerve and initiates the release of zyme activity. Crotoxin is the first presyn- yet known. But it may be that phospholi- acetylcholine and then eventually stops aptic toxin isolated from snake venom. pase A creates a hole in the nerve-end the release. The role of the acidic subunit A is to membrane and Ca2+ flows to the cyto- At the end of the nerve terminal, there guide the toxin to a specific site; then the plasm. As a result, vesicles containing the are many vesicles containing acetylcho- basic subunit B functions as a presynaptic nerve transmitter acetylcholine discharge line. t3-Bungarotoxin (t3-btx) induces the toxin [3]. Each subunit alone is relatively it. The nucleotide-sequence-encoding t3- 2 influx ofCa + to the nerve terminal; this in nontoxic, but combined, the toxicity is btx Archain has been determined [16]. greatly enhanced [4]. The undissociated Only the B-subunit with phospholipase A2 *Correspondence: Prof. A.T. Tu crotoxin itself shows phospholipase A ac- activity had the ability to release acetyl- Department of Biochemistry and tivity, indicating the active site of subunit choline and the noncatalytic unit of A- Molecular Biology B is not masked by subunit A [5]. Besides subunit has no action [17]. and Colorado State University Yang Chang Fort Collins, Colorado 80523, USA neurotoxicity, subunit B also has hemolyt- [18] reported that phospholipase A2 activ- Tel.: +1970491 1591 ic activity. Subunit B attaches to many ity and presynaptic toxic activity can be Fax: +19704916313 parts of the erythrocyte membranes [6] separated. BIOLOGICALLY ACTIVE AGENTS IN NATURE 57 CHIMIA 52 (1998) Nr. 1/2 (Januar/Februnr) The third type of presynaptic toxin is a tertiary complex of three polypeptide chains. Taipoxin from the venom of the Australian snake, taipan, has three sub- units, a,p, and y, with an Mr of46 000. The number of amino-acid residues present in the subunits is 120, 120, and 135, respec- tively. The a-chain is basic and has phos- pholipase A activity. The fourth type is a quaternary com- plex of four polypeptide chains. Textilo- toxin isolated from Pseudonaja textilis consists of A, B, C, and D subunits. Sub- unit D consists of two identical covalently linked polypeptide chains [l9a-c]. The last type is a single polypeptide- chain presynaptic neurotoxin. An exam- ple of this is notexin from the venom of Notechis scutulatus scutulatus, consisting of 119 amino-acid residues with seven Fig. 1. The role of AChR in muscle depolarization disulfide bonds. It has an Mr of 13400. Notexin has isotoxins; they differ in only muscular junction at the postsynaptic site suddenly released from the vesicle at the one amino-acid residue among the two by combining with acetylcholine receptor end of the nerve. Acetylcholine moves isotoxins [20]. The three-dimensional (AChR). These neurotoxins act on the across the synaptic crevice and reaches the structure of notexin was determined by muscle side, rather than the nerve side. acetylcholine receptor in the muscle. The crystallography. The core of the protein is The so-labeled postsynaptic neurotoxins AChR is composed of five subunits, a, a, very similar to other phospholipases A. are really the toxins affecting the particu- 13, y, 8. When two molecules of acetylcho- The difference, however, exists mainly in lar site of the muscle and should not have line attach to the a-subunits, the AChR the area of residues 56-80 and 85-89 [21]. been designated neurotoxins. Actually, changes configuration and becomes an Not every presynaptic toxin is identi- postsynaptic neurotoxins bind to the ace- open ion channel, permitting certain ions cal in relation to the release of acetylcho- tylcholine receptor in the muscle that is to to pass through (Fig. 1). By this mecha- line from the presynaptic site. With ~btx, receive the neurotransmitter acetylcholine. nism, the depolarization wave reaches the there is an initial burst of acetylcholine, On the other hand, the attachment of ace- muscle. but eventually the release is stopped. Even tylcholine to the acetylcholine receptor is The structure of postsynaptic neuro- though toxins may behave like ~btx, the considered a part of the nerve-transmitter toxins is well studied. There are actually length of time for acetylcholine release is mechanism. From this functional view- two types of these neurotoxins (Fig. 2, A, different for each toxin. Some presynaptic point, it is not unreasonable to call them and C). One type has four disulfide bonds toxins do not release acetylcholine from postsynaptic neurotoxins because of their (called type I or ShOli-chain neurotoxins). the beginning and simply stop the release.