Structure and pharmacology of elapid cytotoxins

Pharmac.Ther.Vol. 36, pp. I to 40, 1988 Printed in Great Britain

Specialist Subject Editor: A .

0163-7258/88 $0.00+0.50 1987 Pergamon Journals Ltd

L. HARVEY

STRUCTURE

AND PHARMACOLOGY

OF ELAPID

CYTOTOXINS M. J. DUFTON* and R. C. HIDERt *Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, U.K. ~fDepartment of Chemistry, University of Essex, Colchester, U.K.

1. INTRODUCTION Elapid snakes of the species Naja (the cobras) and Hemachatus (the ringhals) have extremely potent venoms with which to subdue their prey. The objective is not merely to kill the prey, but to do so at a speed and in a fashion that most suits the snake, especially with regard to the digestive processes that follow. Foremost amongst the toxic principles in their venoms are members of a protein family typified by a chain of length of 60-70 amino acids and a common folding pattern (Dufton and Hider, 1983). The most individually lethal and best studied variants are the postsynaptically active neurotoxins, which are to be found throughout the elapid snakes, whether Asian, African or Australian (Karlsson, 1979). The cytotoxins, however, are variants known only from the cobras and ringhals, and much remains to be understood about their mode of action. Their existence alongside the neurotoxins came to light early this century when cobra venom was noted to be exceptional amongst elapid venoms in having cardiotoxic properties (Elliot, 1905). In 1942, the first attempt was made to isolate the responsible factor (Sarkar et al., 1942) and this has been followed, particularly since the mid-1960's, by a number of independent isolations that vary in both the species of cobra studied and in the purity achieved. Despite the application of a wide range of physiochemical, biochemical and pharmacological methods to these cytotoxin-containing fractions, uncertainty as to the exact modus operandi continues. This uncertainty is reflected in the names other than "cytotoxin" which are used to identify these toxins. Terms such as "Direct Lytic Factor" and "Membrane Toxin" have been devised by those who consider these toxins to act primarily on the membrane lipid bilayer, while "cardiotoxin" implies that a more specific target is intended. We adopt the name "cytotoxin" as the most suitable because it does not define a particular target or presuppose the manner in which the toxicity is achieved. In view of the clear structural homology that exists between the neurotoxins and cytotoxins (Dufton and Hider, 1983), it is notable that few of the current theories of cytotoxin action are in any way compatible with the widely accepted concept of neurotoxin action. The purpose of this review is to describe, as far as is possible, the conformational and physiological properties of the cytotoxins and to ascertain whether the structural homology with the neurotoxins reflects any similarity in mode of action. 2. ISOLATION AND PURIFICATION OF CYTOTOXINS Between the years 1900 and 1940, most of the cobra venom research was centered on physiological studies, but in 1942 B. B. Sarkar and his co-workers achieved the first separation of the component proteins. Using a precipitation method, they isolated neurotoxin-rich and hemolysin-rich fractions from Indian cobra venom (Sarkar et al., 1942). Since neither of these fractions were able to inhibit perfused toad heart activity, Sarkar suggested that there was an additional factor which could produce the heart and circulatory failure seen when whole venom was applied. Several years later, N. K. Sarkar JPT 36/1--A

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confirmed this result and developed an improved isolation procedure based on sequential precipitation with Na2SO4 and NaC1 (Sarkar, 1947). One of the fractions obtained was tested for its ability to arrest toad and cat hearts and was found to have 15 times the potency of whole venom. This material, which lacked hemolytic activity, was termed "cardiotoxin". Subsequently, Ravdonat and Holler (1958) demonstrated the "cardiotoxin" to be a mixture of at least 4 fractions which could be separated by paper chromatography. With the advent of gel-based chromatographic methods (Peterson and Sobel, 1956, and Porath and Flodin, 1959) further purification of venom proteins proceeded apace. Initially, attention was focused on the post-synaptic neurotoxins (Karlsson, 1979), but it soon became apparent that the methods used to purify the neurotoxins sometimes simultaneously produced almost pure cytotoxin fractions. The first cytotoxin preparations sufficiently pure for sequence determination were reported by Narita and Lee (1970), Hayashi et al. (1971), Weise et al. (1973), and Fryklund and Eaker (1973) for Naja naja atra, Naja haje, Naja haje annulifera, and Hemachatus haemachates respectively. Venom purifications have now become standardised and depend on an initial gel permeation step (Sephadex G50 or G75) (Fig. 1) followed by resolution on cation exchange media (carboxymethylcellulose, sulphonyl-propyl Sephadex or BioRex 70 (Amberlite IRC-50)) (Fig. 1). Many cobra venoms contain a number of extremely similar isotoxins, some differing by only a single substitution (e.g. Leu/Isoleu, Ala/Val, Lys/Asn). Some of these are difficult, if not impossible, to separate by ion exchange chromatography and their existence has in many cases only been inferred from sequence studies. In contrast, reverse phase high performance liquid chromatography (RP-HPLC) does possess sufficient resolving power to separate such proteins, as was first demonstrated by Inouye et al. (1981) working with insulin analogues. Wu et al. (1982) subsequently resolved Naja naja atra cardiotoxins III and IV (which tend to co-elute on ion exchange columns) using HPLC. Fortunately, elapid venom neurotoxins and cytotoxins are not irreversibly denatured by the non-aqueous conditions necessary for this technique (Boulain et al., 1982; Hodges et al., 1987). RPHPLC has been used successfully to separate the closely related cytotoxins contained in the venoms of Naja melanoleuca, Naja haje annulifera, Hemachatus haemachates and Naja naja siamensis (Hodges et al., 1987). A typical profile is presented in Fig. 1. When used in preparative mode, the eluted toxin fractions can be diluted 10 fold with water and either lyophilised or dialysed against water. Unfortunately, unlike smaller peptides, the retention characteristics of cytotoxins cannot be predicted from their hydrophobic amino acid content, so optimal conditions have to be established for each cytotoxin variant. Presumably this is because an appreciable proportion of their hydrophobic side chains is not exposed on the external surface of the molecule. Recently, a method has been introduced whereby it is possible to fractionate a large proportion of the polypeptide components of elapid snake venoms in a single step analytical mode (Bougis et al., 1986; Fig. 2). This extremely useful advance enables the investigator to compare venoms from different suppliers, regions of the world and snakes of different ages. In summary, the essential stages of cytotoxin isolation are depicted in Fig. 1. Separation of the venom components is best achieved in the following sequence: by size (gel permeation), by charge (ion exchange), and by hydrophobicity (RP-HPLC). 2.1. REMOVAL OF PHOSPHOLIPASE CONTAMINATION

Although the above procedures will produce relatively pure neurotoxins, suitable even for IH N M R studies (Steinmetz et al., 1981), cytotoxins tend to co-elute with phospholipase A2 during gel chromatography. Normally, contamination is in the range 0.1-0.5% by weight, but on occasions it can be of the order of 5%. Careful gel permeation chromatography on large columns (G50, 2.5 x 120 cm) will, in our experience, minimise the problem, but not abolish it. The presence of even trace amounts of phospholipase A2 is potentially serious for many of the contemporary assays for cytotoxins and so investigators should strive to remove as much as possible.

4

M.J. DtwroN and R. C. HIDER

The reason for the co-elution of phospholipase A 2 (M.W. 13,500) with the cytotoxin (M.W. 7,000) after gel filtration in sodium chloride (150 raM) was reported by Visser and Louw (1977) to be a consequence of a hydrophobic interaction between the enzyme and the gel matrix. In urea (8 M) this interaction is minimized and cytotoxin fractions can be obtained with only traces of phospholipase (<0.001% by weight). Similar results are found when the proteins are eluted with sodium chloride (150 mM) saturated with DLphenylalanine (80 mM) (Visser and Louw, 1977). The disadvantage of both these methods is the difficulty in monitoring protein elution and the management of the saturated solutions. More direct methods based on hydrophobic interactions, immunoaffinity and reverse phase chromatography, are easier to manipulate. Hydrophobic chromatography based on phenyl Sepharose CL-4B was introduced by Louw and Carlsson (1979) and involves application of the contaminated cytotoxin sample to a column equilibrated with ammonium sulphate (2 M). The cytotoxin is desorbed by running a gradient of decreasing ammonium sulphate concentration through the column, elution of the toxin occurring in the region of 0.1-1.0 M. The phospholipase is retained under these conditions and requires urea (8 M) in ammonium acetate (0.05 M, pH 5.5) for removal. High flow rates (> 10 ml hr -m) tend to aid phospholipase elution and therefore should be avoided. The method has been applied to cytotoxins from Naja melanoleuca, Hemachatus haemachates (Louw and Carlsson, 1979) and Naja naja siamensis (Hider and Khader, 1982), and has reduced phospholipase contamination to between 0.005 and 0.001% by weight. Delori and Tessier (1980) produced a monospecific antiserum to a pure phospholipase A 2 from Naja mossambica mossambica venom and applied it to a Sepharose 4B column to which phospholipase A2 had been attached. The specific antibodies retained on the column were eluted under acid conditions, dialysed, and subsequently conjugated to Sepharose 4B. Permeation of contaminated cytotoxin samples through this gel led to a reduction in phospholipase A2 content to between 0.003 and 0.001% by weight. Clearly, this is a highly efficient method despite its requirement for specialized column preparation. A simpler, but nonetheless effective method developed by Mangola, is dependent on phospholipase A2 being amongst the most immunogenic components of snake venom. Mangola (1979), and subsequently Hider and Khader (1982) used antivenom prepared against the venom of the viperid Echis carinatus. The use of antivenom-loaded Sepharose 4B columns considerably enhances the purity of Naja nigricollis and Naja naja siamensis

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Elapid cytotoxins

5

cytotoxins (<0.001% by weight of phospholipase A2). However, prolonged use leads to some leaching of peptides from the affinity columns. RP-HPLC i~ also extremely effective at removing phospholipase A: contamination (Hodges et aL, 1987), radial compression units being capable of use in semi-preparative mode. Cytotoxins purified in this manner are estimated to have a level of phospholipase A 2 contaminatioS below 0.001% by weight. The particular advantages of this method are its speed and high yields, which are typically greater than 90%. When reporting low levels of phospholipase A2 contamination in cytotoxin samples, the method of assay and the specific activity of the contaminating enzyme must be considered. All assays should be run at lipid substrate le;cels well above the critical micellar concentration (CMC), otherwise falsely low estimations of phospholipase activity can result. Suitable phospholipase assays are the pH stat method using egg lipoprotein as substrate (Nieuwenhuizen et aL, 1985), the hydrolysis of 14C-labeled phospholipid (Kramer et al., 1978), the hydrolysis of fluorescent phospholipids (Thur6n et al., 1986, Bougis et al., 1986), and the inhibition of ouabain sensitive (Na +, K +) ATPase activity (Khelif et al., 1985). Regarding specific activity, elapid venom phospholipases have values in the range 100 to 1000 #moles min ~mg protein 1 (Lee and Ho, 1982). Since the exact nature of the contaminating enzyme is rarely known, the values of percentage contamination are inevitably somewhat arbitary. In our laboratory, a value of 500/lmoles min- 1mg protein-' has been selected for all such calculations. 2.2. LYOPHILIZATIONOF CYTOTOXINS Like neurotoxins, cytotoxins can be denatured by freeze-drying, some forming aggregates (E. Karlsson and F. H. H. Carlsson, personal communications). Ammonium acetate is the buffer of choice in toxin isolations because traces can be removed by sublimation. However, ammonium salts trigger cytotoxin denaturation when the concentration becomes sufficiently high during the lyophilization process to thaw the lyophilizate. Once this occurs, the toxin is subjected to acid conditions, ammonia being more volatile than acetic acid. A similar proviso applies to the use of 0.1 M acetic acid. In general, it is a good policy to adjust the toxin solution to pH 7.0 and dialyse against water before freeze-drying. "Spectropore" with its smaller pore size is ideally suited to such work, although "Visking" tubing is adequate for dialysis times of up to 24 hr. When dialysed in this fashion the solid is quite stable for periods of up to 6 months when stored at either - 2 0 ° or +4°C. Frozen solutions of cytotoxins prepared in this way are also stable at - 2 0 ° C for periods up to 6 months, as judged by amino acid analysis, HPLC profiles, and biological activity. 3. CYTOTOXIN SEQUENCE INFORMATION 3.1. GENERAl CLASSIFICATION Although the roles and modes of action of the cytotoxins are somewhat obscure, there is an abundance of information concerning their amino acid sequences, with over 50 variants having been characterized (Table 1) (Mebs, 1985). Generally, the homology between them is considerable, but there are several ways of identifying particular subgroups. Starting with major characters, 11 examples can be separated on the basis of polypeptide chain length (i.e. an extra residue between the first two half-cystines) and the possession of distinct tri-residue sequences between the fourth and fifth half-cystines (i.e. AAT, ADA, TDT and TDA). These sequences have been described as "cytotoxin homologues" (Dufton and Hider, 1983) and they have been marshalled into sub-group E in Table 1. The group is itself heterogeneous in the major aspect of chain length because some of its members have up to 2 additional residues.

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Examination of the remaining 41 sequences shows that the high degree of homology is broken in two major segments of the polypeptide chain, namely between the first and second half cystines (residues 10-12) and between the third and fourth (residues 29-32). When attempts are made to group the cytotoxins according to sequence similarity in these two regions, quite different classifications result depending on which of the two variable sequence segments is given prominence. Thus, grouping according to similarity at residue positions 10, 11 and 12 effectively scrambles the sequence subgroups at positions 29-32 and vice versa. Since there are basically two versions of each variable sequence segment, the most appropriate classification is to construct 4 subclasses (i.e. A, B, C and D) according to the four combinatorial possibilities (Table 1). A useful way to summarize the classification is to consider the presence or absence of proline residues in the two segments. Thus, group A cytotoxins have proline 10 and proline 31; group b has proline 31 but not proline 10; group C has proline 10 but not proline 31, and group D has neither proline 10 or proline 31. According to the present data, no single snake species has all five types of cytotoxin, but Naja haje annulifera does contain types A, C, D and E. Grouping according to the zoological classification of the snake species does not significantly aid rationalization of the sequence data because several different types of cytotoxin can co-exist in the venom of a single snake (i.e. duplications of the cytotoxin gene have occurred). A further complication in this respect is that many of the venom samples from which cytotoxins have been purified are actually pooled milkings from several different individuals. Should the rate of cytotoxin sequence evolution be high and species differences subtle, impressions of the actual cytotoxin complement in any given snake could be misleading. 3.2. EVOLUTION

As well as providing structural information, the cytotoxin sequences also reveal aspects of their evolutionary development and ancestry. In this respect, the sequences provide a major clue to the understanding of cytotoxicity, for they demonstrate clearly that the cytotoxins belong to the same protein family as the co-occurring ~-neurotoxins. Indeed, without the sequence data, it would not have been suspected that cytotoxins and ~neurotoxins are related to each other because their pharmacological properties are so distinct. The nature of the homology with the ~-neurotoxins has been fully described elsewhere (Low, 1979; Dufton and Hider, 1983) but suffice it to say that it essentially concerns those amino acids with important conformational roles. When such a degree of homology is seen, it is highly probably that their basic tertiary structures and even their modes of action are similar. The usual manner in which evolutionary information is obtained from amino acid sequences is by systematically comparing all the sequences with each other and then building an evolutionary tree based on the relative differences found. For the cytotoxins, any such comparison will depend heavily on the two variable segments previously noted (i.e. 10-12 and 29-32). However, the difficulties encountered in finding a suitable tabular classification emphasize the inappropriateness of conventional tree-building methods. The problem is that many parallel substitutions appear to have taken place in the evolutionary development of the two variable segments. This means that whenever two cytotoxin sequences are compared, the number of observed sequence changes is probably much less than the number that have actually taken place since divergence from a common ancestor. Parallelism is a problem in all analyses of protein evolution, but with the cytotoxins, it is a dominating factor (necessitating the "combinatorial-type" classification of Table 1) (Breckenridge and Dufton, 1987). Various attempts have been made to describe the evolution of the cytotoxins with regard to all the other types of toxin (especially the ~-neurotoxins) which show amino acid homology (Hseu et al., 1977; Dayhoff, 1979; Strydom, 1979). While the conventional treebuilding methods yield an evolutionary history for the ~-neurotoxins that is in good agreement with morphological data, the relationship between the major toxin types is not

Elapid cytotoxins

9

clearly defined. Therefore, it is not clear how cytotoxicity as a mode of action is related to neurotoxicity. A different type of analysis, which compares the number and location of chain insertions/deletions, has been applied to the entire family of snake toxins (Dufton, 1984). Contrary to the result obtained by comparing similarities in residue character, the cytotoxins were shown to be most allied (in terms of conformation) to the "short" ~tneurotoxins. In turn, these two groups appeared to be related via a common ancestor to the "long" ~t-neurotoxins. The implication of this version of the evolutionary history is that the cytotoxins are developments of the ct-neurotoxins, and therefore the mode of action of the former could be derived from that of the latter. This view is supported by the limited occurrence of the cytotoxins; whereas ct-neurotoxins are present throughout the African, Asian and Australian elapids, the cytotoxins are limited to only the species Naja and Hemachatus. It has also been reported that significant structural homology exists between the toxin family as a whole and other types of protein (Strydom, 1977; Drenth et al., 1980). In particular, it has been proposed that cytotoxins and phospholipases A2 are related, and therefore that cytotoxins are the ancestors of the 0t-neurotoxins (Strydom, 1979). Owing to the general complexity of evolutionary arguments, these conclusions must be regarded as opinions rather than fact until such time as detailed structural analyses and immuno cross-reactivity data are obtained. 4. CYTOTOXIN CONFORMATION The nature of the sequence resemblance between the cytotoxins and the 0t-neurotoxins leaves little doubt that the tertiary structure of cytotoxins is based on the folding pattern common to the x-ray derived structures of erabutoxin b (Bourne et al., 1985) and 0tcobratoxin (Walkinshaw et al., 1980). Indeed, preliminary reports on the crystallographic analysis of Naja mossambica mossambica Vn4 cytotoxin support this supposition (Fischer et al., 1978; Wang and Yang, 1981; Oimatov et al., 1981). What is not so apparent, however, is the behavior and stability of cytotoxin conformation relative to the neurotoxins under physiological conditions. This is a particularly important consideration, because it is known that for neurotoxins the tertiary structure provides scope for defined conformational changes in response to changes in the environment. Thus, even when more detailed crystallographic data becomes available, emphasis will still be placed on spectroscopic techniques like N M R and CD to reveal the dynamic and allosteric properties of the cytotoxins. The data on cytotoxin conformation and stability has been reviewed elsewhere, but the major findings of N M R and CD are given in Sections 4.1 and 4.2. 4.1. IH NUCLEAR MAGNETIC RESONANCE 2D N M R has verified that the triple stranded fl-sheet fundamental to the known crystal structures of ~-neurotoxins also occurs in cytotoxins (Steinmetz et al., 1981). This had been expected from an examination of the amino acid sequence homology and the outcome of secondary structure prediction methods (Dufton and Hider, 1977; Menez et al., 1978). Although this section of fl-sheet comprises 27% of the polypeptide chain, sequential N M R connectivity patterns demonstrate that a further 25% of the chain is also in the form of extended fl-structure (Hosur et al., 1983). Since the 11-13 segment exhibits slow H/D exchange of its amide NH protons, the additional fl-sheet may involve the first major loop of the cytotoxin molecule (Fig. 3). Turning to specific side chain environments, NOE experiments have shown that tyrosines 23 and 53, methionine 25 and isoleucine 41 can form a closely packed hydrophobic cluster on one face of the triple stranded fl-sheet (Steinmetz et al., 1981). This is confirmed by the high pK values of the tyrosines (Tsetlin et al., 1975; Lauterwein et al., 1978) and their inaccessibility to chemical modification (Keung et al., 1975; Hung et al., 1978; Grognet et al., 1986). The C-terminal chain segment lies on the opposite face of the fl-sheet and

10

M.J. DUFTONand R. C. HIDER

is stabilized by a salt bridge between the terminal ~-carboxyl group and the side chain o f arginine 38. This structural arrangement has also been detected in the "short" neurotoxins (Low, 1979).

4.2. CIRCULAR DICHROISM Circular dichroism spectra are often diagnostic of the secondary structure content of small proteins, and on this basis, they show the cytotoxins to be divisible into two broad groups. One group generates CD spectra which resemble those obtained for "short" neurotoxins (Dufton and Hider, 1983) save that a positive 228 nm band is lacking (Fig. 4). Members of the other group feature a weaker CD absorption between 192 and 195 nm, but do possess a positive 228 nm band (Fig. 4) (Grognet, 1984; Grognet, J., M6nez, A. and Hider, R., unpublished data 1987). This means that the latter spectra more closely resemble those of the "long" neurotoxins (Dufton and Hider, 1983). The differences in CD relate to differences in the conformations adopted by the polypeptide backbones, but in view o f the known crystal structures of representative "short" and "long" neurotoxins, these structural differences need not be large. Indeed, the differences between the two CD classes are most probably associated with different degrees of E-sheet curvature (Illangasekare and Woody, 1986). If the type of cytotoxin CD spectrum is matched to the appropriate amino acid sequences, only 2 positions differ consistently between the two groups, and these are 10 and 31 (Table 1). These two residues are sited in the two regions with the greatest scope for conformational variation, namely segments 10-12 and 29-32. Not only are most of the sequence changes concentrated in these areas, but also proline

FIG. 3. Schematic structure of the cytotoxin backbone. This structure is based on preliminary mHnmr (Steinmetzet aL, 1981;Hosur et al., 1983) and x-ray diffractiondata (Fischer et al., 1978). The residue numbers correspond to those in Table 1. The t-sheet sections are indicated by a dashed line representation of the hydrogen bonds. The two hydrogen bonds introduced in the vicinity of residue 30 are more tentative, but two such bonds exist in the homologous short neurotoxin structures. Invariant residues are representedby the single letter amino acid code. (See Table 1). Residues which are identical in over 66% of the cytotoxin series are indicated by Q. Positions 10, 31 and 54 are indicated by [ ] ; these residues have a key structural role.

Elapid cytotoxins

11

can be present or absent in both. The unique stereochemical properties of this imino acid give rise to the strong expectation that significant changes in local chain geometry accrue from its presence or absence. Indeed, this can be appreciated by considering the Chou and Fasman secondary structure predictions for the cytotoxins (Fig. 5) where the major differences between group A cytotoxins and those in groups B, C and D are centered in the region of residues 10 and 31. The group A toxins possess a higher reverse turn potential in the region 9-12 but a lower potential in the region 28-32. The fl-sheet and ~t-helical potentials for the segment 27-34 are also different for the two groupings. A further difference between the two groups occurs in the region 50-56, where the enhanced helical potential is associated with the presence or absence of methionine 54. The intensity of the circular dichroism at 190 nm is typical of "short" neurotoxins for group A and of "long" neurotoxins for the combined groups B, C and D. This suggests that the sequence combination in which proline is present simultaneously at positions 10 and 31 might be responsible for producing a conformational difference. Since subgroups B, C and D show little CD variation with respect to each other, the actual CD contributions from the 10-12 and 2932 segments do not appear to be entirely responsible. Instead, some combined influence of these segments, such as on the overall curvature of the fl-sheet for example, would be a more probable cause. The CD information, like that obtained by NMR, emphasizes similarities to the conformational properties of the neurotoxins rather than contrasts. Therefore, although there is conformational variation amongst the cytotoxins, they do not appear to explore structural balances outside the ranges already established for the neurotoxins (Dufton and Hider, 1983). Like at-neurotoxins, the cytotoxins are remarkably resistant to denaturation in nonaqueous solvents (Tsetlin et al., 1975; Hung and Chen, 1977; Galat et al., 1985). However, their backbone structure is more sensitive to changes in temperature, generally melting below 70°C (many neurotoxins are stable above 85°C) (Dufton and Hider, 1983). 4.3. ACCESSIBILITYOF AROMATICRESIDUES The phenolic PKa values of both tyrosines 23 and 53 are in the region of 12 (N. mossambica mossambica II, Steinmetz et al. 1981; N. mossambica mossambica IV, Lauterwein et al., 1978; and N. oxiana, Tsetlin et al., 1975), which suggests inaccessibility to the solvent and/or participation in hydrogen bond formation. The restricted environment of the two residues is confirmed by chemical modification (Keung et al., 1975; Hung et al., 1978) and photo-labelling studies (Muskat et al., 1984). It should be noted that chemical

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FIG.4. Circulardichroism spectra of cytotoxins. --, Meanspectra of cytotoxins from group A (1A, 12A, 13A, 14A and 15A, Table 1); - - - ,

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(18B, 20B,25C,29D, 32D, 33D, 34D, 35D, 39D, 41D, Table 1) (Grognet,J., Menez,A. and Hider R., unpublished data).

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FIG. 5. Secondary structure analysis of cytotoxins. - - , Mean prediction for the entire group of cytotoxins (IA-41D); - - - , mean prediction for Group A type cytotoxins (IA, 2A, 3A, 4A, 5A, IlA, 12A, 13A, 14A, 15A); . . . . , mean prediction for Groups B and C and D type cytotoxins (16B, 17B, 18B, 19B, 21C, 22C, 23C, 24C, 25C, 26C, 27C, 28C, 30D, 31D, 32D, 33D, 34D, 37D, 38D, 39D, 40D, 41D). These results are produced from a modified version of the original Chou and Fasman (1974) method for helix, sheet and reverse turn (Dufton and Hider, 1977) incorporating parameters originally developed by Levitt (1978). The parameters were processed by PROSS PR (Morrison, 1985). Clusters of six residues in the case of helix, or five in the case of sheet, and four residues in the case of turn were deemed to be potential nucleation centers if the product of their parameters was greater than or equal to unity.

derivatization of cytotoxins can be complicated and that the uniform view given above may be an oversimplification (Carlsson, 1980). In complete contrast to these two 'core residues' tyrosine-12, when present, is fully exposed to the solvent. For instance, it readily reacts with tetranitromethane (Hung et al., 1978), possesses a phenolic pKa typical of an exposed nonhydrogen-bonded tyrosine and is highly mobile even at low temperatures (Lauterwein et al., 1978). In similar fashion, tryptophan-12 has been demonstrated to be exposed in aqueous solvent by fluorescence studies (Dufourcq and Faucon, 1978). Thus, the extremely hydrophobic first loop must be largely exposed to the solvent. 5. CONFORMATION/SEQUENCE RELATIONSHIPS 5.1. MODELING OF CYTOTOXIN TERTIARY STRUCTURE

In view of both the sequence homology with the ~-neurotoxins and the detailed spectroscopic data, there is good reason to suppose that the cytotoxins have tertiary structures very close to those elucidated for erabutoxin b and 0t-cobratoxin (Dufton and Hider, 1977). Since all the major features of the cytotoxins, such as their disulphide bridge arrangement and loop sizes, are present in either or both of the neurotoxin crystal structures, it has been possible to generate a three-dimensional cytotoxin structure which is capable of explaining known spectroscopic data. This was achieved by a superimposition of the erabutoxin b and ~-cobratoxin tertiary structures on the basis of the triple-stranded #-sheet that both have across their second and third loops. By tracing the two outer loops of ~-cobratoxin and connecting these with the central loop of erabutoxin b, a precise

Elapid cytotoxins

13

chain-length mimic of a typical cytotoxin is obtained (Breckenridge and Dufton Subsequently, Ravdonat and Holler (1958) 1987). Through the use o f molecular graphics, the hypothetical structure was rotated to produce the view shown in Fig. 6. As is evident even between the two independent determinations of the crystal structure of erabutoxin b the extremities of this type of molecule are prone to vary in conformation. This is likely to be especially true of the 29-32 segment of the cytotoxins given its exposed nature and sequence variability. Nevertheless, it should also be pointed out that the constraints placed on cytotoxin conformation by the disulphide bridges and the fundamental triplestranded fl-sheet are considerable, and any deviations from Fig. 6 are anticipated to be small. A low resolution structure of Naja mossambica mossambica cytotoxin III (or Vl~4), which has been the subject of a preliminary report, has a very similar folding pattern to that presented in Fig. 6 (Fischer et al., 1978). 5.2. LOCATIONOF INVARIANT RESIDUES Figure 3 is a schematic representation of the predicted tertiary structure showing both the expected hydrogen bonding and the invariant amino acid positions. The nature of many of the conserved amino acids (i.e. proline, glycine and half-cystine), and their tendency to be clustered in the central area of the molecule, signifies that preservation of molecular shape has been a ruling evolutionary factor. In other words, a particular threedimensional structure is a prerequisite for the functioning of the cytotoxins. This could mean that the cytotoxin binds to a target which requires it to have a specific shape, or that the cytotoxin is required to have defined allosteric responses in the presence of its target. It should, perhaps, be emphasized that where amino acids provide a structural 'skeleton' in a molecule, the 'skeleton' can be both articulated and flexible as well as rigid, and may be capable of concerted responses. 5.3. LOCATIONOF VARIABLE RESIDUES Figure 6 is the a-carbon skeleton of a typical cytotoxin as predicted by molecular graphics. It displays those residue positions which are not conserved in at least 66% o f the

\ @ 10

so

.i

FIG. 6. Backbone structure of typical cytotoxin. The backbone has been derived, by the use of moleculargraphics, from the combinedX-ray coordinates of a short neurotoxin,erabutoxinb, and a long neurotoxin, ct-cobratoxin(Breckenridgeand Dufton, 1987). Residues which are not conserved in at least 66% of the known sequences are represented as shaded circles.

14

M . J . DUFTON and R. C. HIDER

known cytotoxin sequences. The locations of these residues highlight the loop extremities and other exposed segments so in general they are disposed over the surface of the molecule. Clearly, on binding to a target, exterior residues of the cytotoxin will be encountered first, so cytotoxin variants can present very different surfaces even though a majority of their structure is constant. The consequence of this variability can be seen in the range of efficacies found for cytotoxins in depolarization assays (see Section 7). As already described, the residue section 29-32 is the most variable part of the primary structure of cytotoxins and it can be seen from Figs 3 and 6 that this section forms the major extremity of the molecule. The other variable section mentioned, namely 10-12, also forms an extremity involving the first major loop. Another interesting feature of the variable positions is that some have apparently evolved interdependently; that is, a change in residue character at one position seems to have coincided with a residue change at another. Positions which behave in this fashion are frequently quite removed from one another in sequential terms but turn out to be spatially proximate in the three-dimensional structure (e.g. the groups 10/12/60, 26/28/51 and 29/31/32 (Fig. 7)). The interdependent relationships between these sidechain positions may be accounted for in two ways. On the one hand, intramolecular dictates of chain folding and side chain packing could set limits to the residue combinations acceptable in the areas involved. On the other hand, there might be intermolecular evolutionary constraints (originating from the target, for example) which dictate that only certain configurations are suited in these areas as regards the process of recognition. It is difficult to resolve which case applies, but molecular packing may be the major factor in the areas involving residues 10, 12 and 60, and residues 26, 28 and 51. In contrast, the variation at positions 29, 31 and 32 is not so readily explained in terms of molecular packing and so a functional criterion may be uppermost. The two types of evolutionary constraint need not be mutually exclusive, however, and all the side chain variations may have varying degrees of structure/function significance.

FIO. 7. Variable residues on the cytotoxin backbone. Location of residue positions which appear to have evolved interdependently. The clusters of positions connected by arrows have changed more or less in concert with each other during evolution (Table 1) and may therefore be subject to a mutual constraint.

Elapid cytotoxins

15

6. M O D E OF A C T I O N OF C Y T O T O X I N S 6.1. NATURE OF BINDING SITE By virtue of their ability to lyse a wide variety of cells (e.g. erythrocytes, fibroblasts, tumor cells and liposomes) comparisons have been prompted between the cytotoxins and other types of toxin which appear to be detergent-like and rather nonspecific in their action. Certainly, the conserved basic/hydrophobic character of the cytotoxins mirrors the other lytic toxins, but there is evidence that the interaction of cytotoxins with membranes is more than a simple lipid/water partition problem. Fibroblast and skeletal muscle membranes, for example, appear to have distinct binding sites (Lin Shiau et al., 1976) and 125Ilabelled cytotoxins can bind strongly to erythrocytes at sub-lytic concentrations (Schroeter et al., 1973). Accordingly, while lipid-cytotoxin interactions require consideration, so do interactions with protein and carbohydrate. 6.2. LIPID-CYTOTOXININTERACTIONS Multiple electrostatic interactions can result from the approach of a basic cytotoxin (typical net charge 10+) to an aggregate of negatively charged surfactant molecules (e.g. a micelle or bilayer). Examples of such molecules are sodium dodecylsulphate, diacetyl phosphate, phosphatidylserine and cardiolipin. This type of interaction was first described independently by Dufourcq and Faucon (1978) and Vincent et aL, 0978). A typical result based on fluorescence measurements (Fig. 8) is shown for the interaction between cytotoxin 13A (Table l) and phosphatidylserine. The association constant for this interaction is > 106M-I with a stoichiometry of 7-10 lipids per toxin molecule. The interaction involves the penetration o f the hydrophobic first loop (residues 6-15) into the hydrocarbon phase of the membrane. The apparent molecular area of cytotoxins in negative lipid monolayers depends on the surface pressure, being 14nm 2 below 20 dynes cm -1 and 5 5 nm 2 above 30 dynes cm-1. These two areas are thought to signify different orientations of the protein with respect to the membrane surface (Bougis et aL, 1982). The cytotoxin-lipid interaction is reversed by high Ca 2+ concentrations (Fig. 9), a phenomenon also reported in many pharmacological assays for cytotoxins.

2 -

=3

f I (~)

/

t

i

d

t

~)

o

O

O-

2

I -

o

o 300

%o 38o wavelength (nm)

/O

I I I I I I

0 4 8 19 16 mote phospholipid/ mote cardiofoxin

FIG. 8. Interaction of Naja mossambica mossambica cytotoxin II with phosphatidylserine. The cytotoxin was dissolved at 25°C in a 50 mM Tris-HCl buffer (pH 8.0), containing 1 mM EDTA to inhibit any trace of phospholipase activity. Aliquots of stock solutions of negatively charged phospholipid vesicleswere added and the fluorescenceemission spectrum was recorded immediately after mixing. A. Spectrum of free cardiotoxin (a) and of complexformed betweencytotoxin and phosphatidylserine (b). B. Titration of cytotoxin with phosphatidyl serine. The relative increase in fluorescenceat 333 nm (F-Fo/Fo), is plotted against the molar ratio between negative phospholipid and cardiotoxin. F is the fluorescenceintensity measured at 333 nm for a given amount of negative phospholipid added, F0 is the fluorescenceintensity of cardiotoxin at 333 nm. The titrations were carried out with two different cardiotoxin concentrations: 0.67 #M(O) and 6.7/zM(O). (Redrawn from Vincent et al., 1978).

16

M.J. DUFTONand R. C. HIDER 200

I

I

i

i

\ Ca2

100

,t,..

,

0

Mg 2'

10-/,

10-3

1 ~0-z '. 10-1 concentration[Mm M FIG. 9. Influence of cations on the binding of Naja mossambica mossambica cytotoxin II to phosphatidylserine. The binding is monitored by a change in fluoresenceintensity at 335 nm. Aqueous solutionis bufferedto pH 7.5 (Dufoureqand Faucon, 1978).The approximateEsovalues are: Ca2+, 0.01M;Mg2+, 0.05 ra; K + and Na +, 1 ra

E

I

=0

I

q

m ,0

I

I

PS [- surface ,I. pressure_ T of ~ eryfhrocyfe

~LP[~

membrane _

m. 5

°-\ .\\\-

0

10

20

30

L*O

Inifiat surface pressure (dynes/era)

FIG. 10. Surfacepressure of Naja mossambica mossambica cytotoxinIV in monolayersof different lipids. The change in the maximumvalue of surface pressure was recorded as a function of initial filmpressure. 0, Cytotoxin/dilaurylphosphatidylcholine;O, cytotoxin/dilaurylphosphatidylserine; F-l, melittin/dilaurylphosphatidylcholine;I , melittin/dilaurylphosphatidylserine.[Toxins]= 1 0 - 7 M (redrawn from Bougis et aL, 1981). The binding of cytotoxins to neutral phospholipids is much weaker compared with that to acidic lipids (Vincent et al., 1978; Bougis et al., 1983; Dufourcq et al., 1982). A cytotoxin isolated from N. n. siamensis venom (35D, Table 1) was found to dissolve preferentially in the aqueous subphase of egg yolk phosphatidylcholine at pressures higher than 20 dynes cm -~ (Hider and Khader, 1982). A similar finding has been reported for N. mossambica mossambica cytotoxin IV (14A, Table 1), where a marked contrast was observed for the interaction with dilauryl phosphatidylcholine and dilauryl phosphatidylserine monolayers (Fig. 10). This behavior is quite different from that of the well characterized lytic peptide melittin (Bougis et al., 1983). Significantly, 15%, or less, of the lipids in erythrocyte (Bretscher, 1972; Rothman and Lenard, 1977), fibroblast (Sessions and Horwitz, 1983), and excitable cell plasma membranes (Michaelson et al., 1983) possess a net negative charge, and virtually all of these lipids are located on the cytoplasmic face. Thus, the outer membrane leaflet of these cells will be composed of neutral phospholipids at a surface pressure of approximately 30 dynes cm -~ (Demel et al., 1975). These conditions are unfavorable for an interaction with the cytotoxins (Fig. 8 and 10). In support of this conclusion, liposomes prepared from phosphatidylserine/phosphatidylcholine mixtures (1:4 and 1:19) have been reported to interact with cytotoxins to a much lower extent than when only phosphatidylserine is used. Indeed there is virtually no interaction with liposomes containing 5% phosphatidylserine, as judged by fluorescence and centrifugation studies (Dufourcq and Faucon, 1978). As the distribution of negative phospholipids is not symmetrical in unilamellar liposomes, with preference being shown for the external leaflet (Michaelson et al., 1973), the percentage of these lipids on the outer surface would be expected to be greater than 5%. Thus, even in the unlikely event that the asymmetric

Elapid cytotoxins

17

distribution of negatively charged phospholipids is not complete, their concentration in the outer membrane could not reach the levels required for an appreciable electrostatic interaction with cytotoxins. In apparent contradiction to the above conclusion, cytotoxins have been reported to cause lysis of liposomes prepared from phospholipids possessing zero net charge (Hsia et al., 1978; Chen et al., 1981). However, the degree of phospholipase A 2 contamination of the cytotoxins was not reported and, as a marked synergistic lytic effect with the enzyme exists, it is possible that this contamination is responsible for the reported observations. This interpretation is supported by the finding that lytic activity was dependent on the presence of Ca 2+ (Hsia et al., 1978) in complete contrast to observations with erythrocytes (Zusman et al., 1984). Since there is also little reason to suppose that cytotoxins will interact with glycolipids in a manner different to that of phosphatidylcholine, the initial interaction of cytotoxins with membrane surfaces is unlikely to involve lipids. This conclusion offers a ready explanation for the apparent inability of many highly purified cytotoxins (~< l0/~M) to induce erythrocyte lysis (Bougis et al., 1983; Hodges et al., 1987). 6.3. PROTEIN/CARBOHYDRATE-CYTOTOXININTERACTIONS

Proteins and carbohydrates are integral parts of cell membranes, providing both transmembrane mechanisms and cytoskeletal support. They must be seriously considered as possible cytotoxin binding sites because many of them possess anionic regions in their extracellular segments. The erythrocyte membrane, for example, has a high content of glycophorin, a glycoprotein which is uniformly dispersed by virtue of mutual electrostatic repulsion (Fig. 11). The marked anionic nature of the extracellular portion of glycophorin is in part provided by sialic acid residues in its attached oligosaccharide chains, but there is also a cluster of anionic amino acid residues at the extracellular boundary of its transmembrane segment. Clearly, the general cationic nature of the cytotoxins could have important consequences for the disposition of membrane macromolecules like glycophorin if electrostatic binding occurs. Many types of membrane protein present anionic carbohydrate moieties at the extracellular surface, but there is evidence to show that these are not necessarily crucial to cytotoxin action. Neuraminidase, an enzyme which specifically removes terminal sialic acid residues from oligosaccharide chains, does not alter cytotoxin action on thyroid slices (Wolff et al., 1968), erythrocytes (Condrea et aL, 1971), or atrial muscle (Harvey et al., 1982). While specific protein/cytotoxin interactions are therefore unlikely to be mediated by the carbohydrate moieties, the anionic regions in the polypeptide chains themselves could nevertheless be involved. Moreover, since these latter anionic regions commonly occur at the outer boundary of the lipid bilayer, a proximate hydrophobic environment is present which could also complement the hydrophobic character of the cytotoxins. One of the most widely reported properties of cytotoxins is their ability to act synergistically with phospholipase A2 (Fig. 12). Although there is some doubt as to whether or not cytotoxins have direct lytic properties, there is no question concerning the synergistic activity. Since the synergism can be induced by preincubation with cytotoxin, this suggests that cytotoxins can bind to the erythrocyte surface. The binding of a 12SI-cytotoxin (51 E, Table l) to human erythrocytes has been studied in Tris-buffered saline at 37°C (Condrea et al., 1965). When used at sublytic concentrations, binding to the external face of the membrane can be achieved without the complication of extensive binding to the moieties on the inner face of the membrane. Once bound, it is difficult to dissociate cytotoxins from the cell. Using an entirely different approach, cytotoxins have been bound to specific acceptor molecules in fibroblast membranes, the binding leading to the formation of membrane lesions of limited size (Thelestram and Mollby, 1979). Surprisingly, rather little work has been directed towards the binding of cytotoxins to intact cells, an aspect that deserves more consideration. High affinity binding sites have been demonstrated for n5I-labelled JPT 36/1--B

18

M.J. DuFTONand R. C. HIDER

2 36

1

FIG. 11. Outer surface of human red blood cell. 1, glycophorin A, 2, band 3 protein; 3, globoside, and 4 polylactosamine ceramide. O, hexoses; O, hexoseamines; and e , sialic acid. (Redrawn from Viitala and J[irnefelt, 1985)

I

21, - -

100

i

I

/,/ 8O

&

_

®

~_--

j-zo

I

I

//

--

//

--

/////

2O

-

/ / ~

/

/11

//

--

-

/ / // -

//

,,

~o

I

(~)

m~

6O

c}.

[

// -

//

~ ~_

g 10

lime (rain)

,

Io 20

~

l/ I

I 0.1

I

t

t

0.2

[Erythrocyfe] %

0-3

v/v

FIG. 12. Synergism of hemolysis by cytotoxins and phospholipase A2. Time course of hemolysis. Erythrocytes (0.05%, v/v) were incubated in buffered saline (pH 7.4) containing Ca2+ (0.6 raM), cytotoxin 51E (35 t~gml-'), in the presence ( - - - ) and absence (--) of Naja mossambica mossambica phospholipase A2 (80 ng ml-t). Effect of erythrocyte concentration on hemolysis rates. Rate of lysis determined by monitoring OD700over 10 rain. Erythrocytes were incubated in buffered saline (pH 7.4) containing Ca2+ (0.6 m~), cytotoxin 13A (92 #g ml -l) and in the presence (- - -) and absence (--) of Naja mossambica mossambica pbospholipase A2 (1.5 #g ml -t) (redrawn permission from Louw and Visser, 1978). cytotoxins on chick biventer cervicis muscle (Lin Shiau e t al., 1976) and on h u m a n a m n i o n cells (Takechi e t al., 1986). Thus, o u r conclusion is that the available physicochemical and biochemical data is best interpreted in terms o f cytotoxins being able to bind to a limited n u m b e r o f m e m b r a n e protein sites on the outer face o f cytoplasmic membranes.

Elapid cytotoxins

19

6.4. MECHANISMOF CELL LYSIS Once contaminating phospholipase is removed from the cytotoxins, high concentrations ( > 10-SM) and long incubation times are required to produce lysis of human erythrocytes, as shown in Fig. 13 (Bougis et aL, 1983, Hodges et al., 1987). With some cytotoxins, definite lag periods are observed before the onset of lysis (Fig. 13, Schroeter et al., 1973) and it is this aspect of their behavior which distinguishes them from other well characterized hemolytic agents. Melittin, for example, produces a much more rapid onset of lysis at lower concentrations ( < 10-6M)(Degrado et aL, 1982). Although many lytic agents generate linear Arrhenius plots throughout the temperature range 0°C to 30°C (Irmscher and Jung, 1977) cytotoxins do not, their lytic action ceasing below 15°C (Fig. 13, Schroeter et aL, 1973 and Chen et al., 1984). It is nevertheless possible to calculate the activation energies for cytotoxin-induced lysis for the 20-40°C temperature range (Louw and Visser, 1977). Working on the assumption that the lytic activity is proportional to toxin concentration, the activation energies for two cytotoxins (13A, 51E, Table l) proved to be indistinguishable, namely at 60 kJ tool -1. This value is similar to the activation energy determined for alamethicin-induced erythrocyte lysis which at 62 kJ tool 1 (0_25oc), is larger than the value for melittin (49 kJ tool -1) (Irmscher and Jung, 1977). Melittin differs from the cytotoxins in being able to interact strongly with neutral phospholipids (Fig. 10). Therefore it can rapidly penetrate the outer face of an erythrocyte membrane. Once incorporated in the membrane, melittin can be considered as a molecular "wedge" (Dawson et al., 1978) which alters the lipid-lipid packing, thereby influencing the fluidity and phase separation of the membrane lipids. The link between this initial disruption of the membrane and the onset of lysis is not clear at present (Tosteson et al., 1985 and Dufton et al., 1984). However, as outlined in the previous section, it is unlikely that these first steps are the same for cytotoxin-induced lysis. During the nonlytic period of the cytotoxin-erythrocyte interaction, the erythrocyte membrane is weakened and becomes more susceptible to the effects of hypotonic media (Chen et al., 1984). When human erythrocytes were investigated for osmotic sensitivity, 50% were found to lyse after 4 hr incubation in approximately 80 mu sodium chloride, but lysis was very limited above 90 mM. In contrast, when preincubated with cytotoxin 18B (Table l) for 15 rain at 37 °, 50% hemolysis could be achieved in l l 0 m u sodium chloride, with 25% of cells lysing even at 150 mu (Fig. 14). Significantly, no such effect resulted when the cells were incubated with cytotoxin at 15°C or with phospholipase A 2 at 37°C. Thus, it appears that cytotoxin binding to the erythrocyte membrane leads to the gradual weakening of the cell. Since enhanced rates of osmotic induced lysis indicate an :increase in transmembrane ionic leaks, ion channel formation may be involved. Indeed,

JJg/m[ I

10

100

I

I

1000

T I IITIII

I

N. metano[euca

~25

oO t

I I IIII1~

I

IIIIII

H. haemachafus

1 0

I0-7

I0-6

I0-5

80

160

2/+0

320

10"/*

cardiofoxin [HI FIG. 13. Cytotoxininduced hemolysis. (A) The hemolyticeffectof two cytotoxinsisolated from each of Naja melanoleuca and Hemachatus haemachates venoms on human erythrocytes.N. melanoleuca, <~ Vlll; © VII2; H. haemachates, C)9B; <) 12B. (from Hodges et al., 1987). (B) The influence of temperature on the hemolyticactivityof H. haemachates 12B (from Schroeteret al., 1973.)

20

M . J . DUFTON and R. C. HIDER

when cytotoxins are incubated with erythrocytes at 37 °, swelling is observed in an increasing proportion of the cells with time (Junankar, P. and Wyatt, K; personal communication). This enhanced influx of water is almost certainly related to induced transmembrane ion leaks. In order to prevent erythrocytes swelling as a result of their extremely high hemoglobin concentration, the intracellular ionic strength is normally maintained at a lower level than that of the plasma. This asymmetry is achieved via the Na +, K+-activated ATPase (Fig. 15). The human erythrocyte membrane is freely permeable to anions via at least two protein mediated routes (Passow, 1986; and Hoffmann, 1986), one of which is the anion exchange protein, Band 3. If a nonselective channel is introduced into the membrane which is permeable to both cations and anions (e.g. alamethicin), it will facilitate Na + entry, together with a counter anion, and cause cell swelling (Fig. 15a). A similar sequence of events is predicted to occur following the introduction of a Na+-specific channel, because counter anions can enter the erythrocyte via the endogenous anion translocators. In contrast, neither K + ionophores or anion selective channels will trigger cell swelling. K + efflux will occur with the former, leading to cell shrinkage (Fig. 15b) and with anion selective channels, little change will result as the membrane is already highly permeable to anions by virtue of the two endogenous translocation systems (Fig. 15c). Members of the family of alamethicin molecules produced by the Trichoderma viridae (Pandey et al., 1977) form well-characterized oligomeric transmembrane channels (Nagaraj and Balaram, 1981; Fox and Richards, 1982). Alamethicin itself possesses a single negative charge and therefore oligomeric channels formed from this antibiotic are not highly charged and are permeable to both cations and anions as a result (Hanke and Boheim, 1980). As shown in Fig. 15a, alamethicin is able to trigger erythrocyte lysis (Irmscher and Jung, 1977) but valinomycin, which is a relatively specific K + ionophore, does not (Hunter, 1977). Cytotoxins isolated from both Naja naja siamensis (35D) and Naja oxiana (19B) have been reported to form anion selective oligomeric channels in black lipid membranes (Ksenzhek et al., 1978; Sandblom and Diaz, 1984). This property is very probably associated with their high net positive charge. As indicated in Fig. 15, the creation of anion channels is not predicted to lead to erythrocyte lysis. Thus, although incubation of erythrocytes with cytotoxins produces osmotic fragility (Fig. 14), this is apparently not a result of oligomeric cytotoxin channel formation. In a comparative study of the influence of 38 cytolytic agents on fibroblasts, the cytotoxins were found to give rise to membrane lesions of limited size (Thelestram and Mollby, 1979). The cytotoxins investigated (16B and 51 E, Table 1) enhanced the permeability of fibroblast membranes towards aminoisobutyric acid but not to macromolecules. It was suggested that the cytotoxins interact with a membrane acceptor protein and thereby generate a transmembrane channel of limited dimensions. A pore of sufficient diameter to permit entry of the zwitterionic isoaminobutyric acid is also likely to permit entry of both cations and anions, so this is a plausible explanation for the cytotoxin-induced osmotic sensitivity of erythrocytes.

100 -

d~

",,..\

5O

0

I 0 05

I 007

I 009

0.11

NaCt, (1~)

0.13

JR . 0.15

Fro. 14. Osmotic sensitivityof human erythrocytesin hypotonic saline. Membrane fragility of erythrocytespre-incubatedat 37° for 15 min in plasma extender solutionswith (---) and without (--) 7.5 #M phospholipase A2. Membrane fragility of cardiotoxin-pretreated erythrocytes: preincubation at 37° (----), preincubation at 15° (-.-.). 15 min preincubation and 4 hr incubation (Chert et al., 1984).

21

Elapid cytotoxins

IN ATP

OUT

II

II

.

3Na+

(a

Stable volume L Cetts

c

/b

swet[

H20 F Cetts

shrink

K+

L

H20

r Stabte volume L

Ct-~

Ct-

(c)

(d)

FIG. 15. Transmembrane ion fluxes in erythrocytes. (a) Native state, Na ÷, K+-activated Mg:+dependent ATPase maintains an asymmetric distribution of Na ÷ and K ÷ ions and resulting osmotic balance. (b) Introduction of a nonselective channel leads to influx of ions and a coupled flow of water, so cells swell. (c) Introduction of a K ÷ ionophore leads to K ÷ etflux and cell shrinkage. (d) Introduction of an anion selective channel induces little change as the membrane is already highly permeable towards CI- via the anion exchanger, which is capable of functioning in a non-exchange mode (Passow, 1986). O, Native ion translocators; [], non-native ion translocators. Normal intracellular ion levels (mM), Na ÷, 10; K ÷, 85; CI-, 70; HCOs-, I1; normal extracellular ion levels (mM), Na ÷, 145; K ÷, 4; CI-, 100; HCO3- , 25.

6.5. CYTOTOXIN-INDUCED PROTEIN AGGREGATION

In Section 6.3, arguments were presented for cytotoxins binding to a protein component on the outer face of erythrocyte membranes. Unfortunately, surprisingly few experiments have been designed which involve the measurement of binding of labeled cytotoxins to intact cells. Some experiments have been reported for membrane fragments and erythrocyte ghosts, but as cytotoxins are also probably capable of binding to negatively charged moieties on the intracellular face of the cytoplasmic membrane, the results are not straightforward to interpret. The limited number of cytotoxin binding sites (2-3 x 105 per cell) reported by Condrea et al., (1965) could be provided by either Band 3 protein or glycophorin, which are present at levels of 106 and 5 x 105 copies per cell respectively (Viitala and J/irnefelt, 1985). As indicated earlier (Section 6.3), the carbohydrate moieties of these proteins are probably not involved in the binding process in view of the failure of neuraminidase to interfere with the lytic action. Band 3 protein forms dimers and tetramers in erythrocyte membranes and these oligomers can act as non-specific ion channels in reconstituted systems (Van Hoogevest et al., 1984), presumably by generating aqueous pores between the protein surfaces. There is an anionic region in the external domain of Band 3 protein largely consisting of loop 491-507, which possesses a net negative charge of 4 (Kopito and Lodish, 1985). It is conceivable that the strongly basic cytotoxins could bind to these sites, converting them from a region of hydrophilic character to one of hydrophobic character. This conversion could lead to tetramer and higher oligomer formation (Fig. 16). Oligomerization of Band 3 protein leads to the generation of new transmembrane protein-protein interfaces. If the

22

M.J. DUFTONand R. C. HIDER

packing of these surfaces is not perfect then nonspecific transmembrane pores will be generated (Fig. 16) which will operate in a manner similar to that of the alamethicin oligomeric pore (Fig. 15b). This mechanism readily accounts for both the temperature sensitive nature of cytotoxin-induced hemolysis and the nonlytic lag phase at higher temperatures (Fig. 13b). Below 20°C the translational motion of Band 3 protein is extremely low (Nigg and Cherry, 1979) and therefore the state of oligomer formation would be low. In contrast, melittin and alamethicin, which probably act directly on the lipid bilayer, are capable of inducing rapid lysis at 0°C (Irmscher and Jung, 1977). 6.6. CYTOTOXIN-PHOSPHOLIPASESYNERGISM The lytic synergism between cytotoxins and phospholipase A 2 follows from the cytotoxin-induced changes in the cell membrane. In principle, an enhanced phospholipid "flipflop" rate associated with an increased transmembrane ion flux (Dressier et al., 1983) could be responsible for potentiating the phospholipase action, particularly if the concentration of negatively charged phospholipids in the outer leaflet is increased. However, the native orientation of phosphatidylserine is much more resistant to perturbation than that of phosphatidylethanolamine (Schneider et al., 1986). Thus, it is unlikely that the presence of cytotoxins would trigger a rapid exchange of this phospholipid. An alternative explanation for the synergism based on the aggregation of Band 3 protein is that the cytotoxins could create protein-free areas on the erythrocyte surface. These regions can be expected to be more susceptible to phospholipase binding, simply because of less crowding from the membrane proteins (Fig. 11). 6.7. INTERACTIONOF CYTOTOXINS WITH MUSCLE CELLS As indicated in the introductory sections, cytotoxins are capable of depolarizing muscle cells (Fig. 17) and thereby triggering contraction (Fig. 18) (Lee, 1972; Harvey, 1985; Harvey et al., 1982). Cytotoxins influence cardiac, skeletal and smooth muscle and also induce a loss of excitability in neurons (Lee et al., 1968). In contrast to erythrocytes, these tissues are affected by cytotoxin at levels of 1/~M and there is no marked synergism with

Cytotoxin

1~/ temperature insensitive

1~ temperature sensitive

~

a q u e o u p o e rs

¢

higher otigomers FIG. 16. Hypothetical Band 3 protein oligomeric pore formation induced by cytotoxins.

Elapid cytotoxins

23

phospholipase A2 (Harvey et al., 1983). It seems probable, therefore, that excitable cells possess a cytotoxin-binding protein as part of their plasma membrane which is either not present, or present at a much lower concentration, in erythrocyte membranes. On the basis of several observations on rat cardiac muscle, including selectivity, reversibility and induction of tachyphylaxis, Sun and Walker (1986) have also postulated the existence of a specific binding site for cytotoxins in this tissue. A number of cytotoxin targets have been suggested, including ATP-dependent cation pumps, endogenous phospholipases, membrane bound calcium stores and transmembrane cation channels.

6.7.1. ATP-dependent Cation Pumps Cytotoxins will inhibit Na +, K +- activated ATPase activity in a variety of cells (Zaheer et al., 1975) and so in principle could trigger depolarization by abolishing the electrogenic

influence of the pump. However, the electrogenic component o f the resting membrane potential in both skeletal muscle and nonmyelinated neurones is minimal (Thomas, 1972) 60 ~ESO

CTx N

,°

2pglml

tOIJglml

I

A I 0

I t0

I J l 2@ 30 40 TIME (rain)

' $0

J 60

J 70

FIG. 17. Depolarizationof cultured skeletalmusclefibersby Naja naja siamensis cytotoxinII (35D, Table 1). The influenceof cytotoxin at differentconcentrationson the rate of membrane potential depolarization (reproduced with permission from Harvey et al., 1982). A. CBC

ZX CTxlV B. G p D

A CTxlV

°,. 1 CTxlV 5pg/ml

FIG. 18. Effect of Naja naja siamensis cytotoxin (CTX IV, 35D) on three muscle preparations. (a) Chick biventer cervicis (CBC) nerve muscle preparation (indirect stimulation, 0.1 Hz). (b) Guinea pig hemidiaphragm-phrenic nerve (GpD) preparation (indirect stimulation, 0.1 Hz). (c) Guinea pig left atria (GpA) (stimulation at 2 Hz) (reproducedwith permission from Harvey et al., 1982).

24

M . J . DuvroN and R. C. HIDER TABLE 2 Increase in ~Ca e+ Uptake by Paired Chick Biventer Cervicis

Muscles Induced by the Presence of Naja naja atra Cytotoxin Ill (1.5/ZM) (From Lin Shiau et al., 1976). Modified physiological saline Normal Low Ca 2+ High Ca 2+ High Mg2+

45Ca2+ uptake (% of control) [Cation] mM 10 -3 12 10

-- CTX

+ CTX

100 158+ 18 62+ 16 378 __+20

372_ 24 714__+26 64+23 1453 + 5

and it would not be possible rapidly to depolarize such cells solely by inhibiting the sodium pump. Indeed, specific inhibition of the sodium pump by ouabain has quite different consequences for both skeletal and cardiac muscle compared with cytotoxins (Harvey et al., 1982). Moreover, since not all cytotoxins inhibit Na ÷, K÷-activated Mg2+-dependent ATPase activity (Hider and Harvey, 1982), this mode of action is unlikely. Cytotoxins have also been demonstrated to inhibit Ca 2+, Mg2+-activated ATPase activity in both erythrocytes and muscle tissue (Fourie et al., 1983). Clearly, the resulting elevated intracellular Ca 2+ levels could trigger muscle contraction and simultaneously depolarize the cell membrane via the Ca-dependent ATPase. However, relatively high cytotoxin concentrations are required to cause appreciable inhibition of the enzyme (50% inhibition occurs at 10 -4 M cytotoxin), So it is difficult to envisage that the strong contractions induced by the cytotoxins are the result of nonspecific transmembrane Ca 2+ pathways. 6.7.2. Endogenous Phospholipase A2 The regulation of endogenous phospholipase A2 could in principle be subverted by cytotoxins to induce self-destruction; for instance, in heart and smooth muscle, conversion of phosphatidylcholine to arachidonic acid could initiate contraction via prostaglandin formation (Shier, 1982). Two cytotoxins (18B and 51E, Table 1) have been shown to enhance both endogenous phospholipase activity and prostaglandin synthesis in cultured fibroblasts (Shier, 1980). However the activtion of the phospholipase displays a distinct lag phase of approximately 20 min, which is incompatible with the rapid onset of muscle contraction. Furthermore, such a mechanism is improbable for skeletal muscle or nervous tissue. 6.7.3. Displacement of Membrane Bound Ca e+ Smooth muscle possesses an appreciable store of Ca z+ which is bound to the plasma membrane and it has been proposed that cytotoxins could displace this and thereby trigger contraction. Such a mechanism is unlikely for skeletal muscle and neurons because cytoplasmic membrane Ca 2÷ stores are either small or non-existent. In addition, pretreatment of cardiac muscle with lanthanum (which displaces Ca2+ions), fails to inhibit cytotoxin-induced contractions (Harvey et al., 1982). A further argument against this concept is provided by the detailed experiments on biventer cervicis muscle whereby extracellular, not intracellular, Ca z+ has been implicated in muscle contraction (Harvey et al., 1982). 6.7.4. Activation of Endogenous Ca 2+ Channel Excitable membranes are depolarized as a result of cytotoxin binding and therefore a proportion of the voltage-dependent Ca 2÷ channels will be activated. However, depolarization is not essential for cytotoxin-induced contraction; for instance, cytotoxins can induce contraction in low sodium media (Lin Shiau et al., 1983). This and related findings suggest that cytotoxins directly enhance the permeability of the cytoplasmic membrane towards Ca 2÷. As previously discussed, an oligomeric cytotoxin channel, should it form, is

Elapid cytotoxins

25

unlikely to be freely permeable to cations. Thus, activation of an endogenous Ca 2+ channel would appear to be the most probable mechanism for cytotoxin-induced muscle contraction. That this is a logical development for snake venom components is illustrated by the observation that a toxin present in the venom of the viperid Crotalus atrox behaves as an agonist for voltage-dependent Ca 2÷ channels in guinea-pig heart preparations (Hamilton et al., 1985). Naja naja atra cytotoxin III (18B, Table) has been shown to enhance the rate of 45Ca2+ influx into skeletal muscle, the rate being potentiated by low extracellular Ca z+ levels (10 -6 M) or by the presence of elevated extracellular Mg 2÷ levels (10 mM) (Table 2) (Lin Shiau et al., 1976). In contrast, the 45Ca2+ influx was markedly inhibited by high extracellular Ca 2+ levels (12 mM). The inhibition of binding of lzSI-cytotoxin 18B to intact muscle by Ca 2÷ (10 mM) indicates that Ca 2+ and the cytotoxin compete for the same site. Two studies have reported that cells develop a sensitivity towards cytotoxins during cell differention, in one case with Rous Sarcoma cells (Kaneda et al., 1985) and in the other, with chicken embryo heart cells in culture (Arms and McPheeters, 1975). With both examples, the increased sensitivity seems to be associated with the introduction of a new membrane protein into the cytoplasmic membrane. Whether or not this binding site is associated with a Ca 2÷ gate remains to be directly demonstrated. Voltage sensitive Ca 2+ channels are heterogeneous (Miller, 1985), and many of those present in membranes may not be (Schwartz et al., 1985). It has been suggested that Ca 2÷ channels in both basophils (Mazurek et al., 1984) and presynaptic membranes (Pumplin et al., 1981; Dunant and Israel, 1985) consist of a number of membrane-bound subunits which aggregate to form an active channel. This aggregation process is under tight physiological control. It is now well established that the binding of polypeptide hormones to receptors can lead to a series of protein-protein interactions amongst dynamic cell surface constituents which normally migrate in the plane of the cell membrane (Hollenberg, 1986). Should the presence of cytotoxins enhance the rate of protein aggregation or stabilize the aggregates, then an increased Ca 2+ influx will result. Aggregation of the type indicated in Fig. 16 could account for such a phenomenon. If the intracellular Ca 2+ levels remain elevated, then Ca 2÷ dependent proteolysis (Ishiura, 1981) and prostaglandin synthesis (Llados, 1985) could lead to the many irreversible effects so widely reported for cytotoxin-induced damage (Lee, 1972; Condrea, 1974). The elevation of intracellular calcium is believed to be the final common pathway through which many diverse toxins act (Schanne et al., 1979). 7. STRUCTURE/ACTIVITY ANALYSIS The investigation of structure/activity relationships in the cytotoxins is problematical for two reasons. Firstly, the lack of a readily identifiable target means that the validity of a particular assay can always be questioned. Secondly, the frequent contamination of "pure" cytotoxins with phospholipase A2 leaves doubt as to whether the effects seen are due to the cytotoxin itself or the result of the very efficient synergism. Most of the comparative data for the cytotoxins centers on three assays: erthrocyte lysis, LDs0 values and muscle cell depolarization. 7.1. ERYTHROCYTELYSIS Of the three assays, the hemolysis test is by far the most sensitive to phospholipase A 2 contamination (Louw and Visser, 1978; Harvey et al., 1983). Some workers contend that erythrocytes cannot be regarded as significant cytotoxin targets, but the fact remains that they are an ideal means of demonstrating cytotoxin/phospholipase A 2 synergism. It has been clearly shown that as phospholipase A2 contamination is removed, then so the lytic powers of "pure" cytotoxins diminish (Bougis et al., 1983; Hodges et aL, 1987). This being the case, it appears hazardous to attempt serious structure/activity studies based on the

26

M . J . DLq~TOS and R. C. HIDER

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Elapid cytotoxins

27

existing data. However, the assay could have future application in establishing the structure/activity of the synergistic process by, say, comparing a range of cytotoxin variants against a single phospholipase A2 variant.

7.2. LDs0 As commonly used, the cytotoxin LDs0 test is performed on mice, the routes of delivery being intravenous, intraperitoneal or subcutaneous. The LDs0 value varies according to the manner of administration, so the data shown in Table 3 pertain to the intravenous route only. This table is a compilation of the variable sequence positions for 24 cytotoxins and they are arranged in order of decreasing toxicity. Despite the unsubtle nature of the LDs0 test and the varying susceptibilities of different mouse strains, some structure/activity information is forthcoming. Notable points are as follows: (i) The sequence evolution does produce changes in lethality, with up to a six-fold difference in LDs0 being possible between two variants. Therefore, it is not solely the invariant residues which determine toxicity. (ii) The cytotoxins Naja haje annulifera 4B/4Ba and the cytotoxins Naja haje annulifera 2H/2Ha (aggregate i.v. LDs0 2.6 and 5.9 mg/kg mouse, respectively) differ only in the change Asp to Asn at position 42 (Fig. 3). Asp 42 is present in all but two of the cytotoxins and is unique to this class of toxin. Therefore, the expectation that this residue is important for cytotoxin action appears to be confirmed. In contrast, replacement of Asp 47 in Naja nivea VII1 by Asn (Naja haje annulifera VII1) has no major effect on toxicity (2.9 and 3.0 mg/kg mouse, respectively). Although Asp at this position is again unique to the cytotoxins, 22 variants possess Asn and 20 possess Ser, so plainly the presence of Asp is not essential. (iii) Replacement of Ala at position 16 in Naja haje haje CM-9 by Glu (giving Naja haje haje CM-7) produces little change in toxicity (2.2 and 2.8 mg/kg mouse). Similarly, the presence of Leu or Arg at position 1 in the cytotoxins Naja naja atra II and IV has no effect on toxicity (both 2.1 mg/kg mouse). As both substitutions involve appreciable changes in chemical character, neither positions 1 or 16 seem to be important for the toxic mechanism. (iv) Naja naja siamensis CM6 and Naja nigricollis 7 differ at 16 residue positions, yet these are the two most toxic variants (0.75 mg/kg mouse). This shows that high toxicity can be maintained despite many changes in sequence. Ten differences exist between the former toxin and Naja haje annulifera CM-2e, yet in this case toxicity changes by a factor of 6.5. Hence, it is not so much the total amount of change that matters, but rather the specific changes therein. Although more intricate structure/activity relationships are identifiable in the LDs0 data, the difficulty in using "death" as an assay is that it can have several contributory factors and their order of precedence may change as regards the final coup de grace. In other words, since cytotoxins have a range of effects, the assumption that death follows from the same aspect of their action in all cases (as is implicit in structure/activity analyses) may be unwarranted.

7.3. MUSCLE CELL DEPOLARIZATION This assay is arguably superior to the others currently available because it is not particularly sensitive to phospholipase A: contamination and involves a specific tissue preparation (Harvey et al., 1983). Only small amounts of toxin are required and readily quantifiable results are obtained. Twenty one cytotoxin variants have been tested and up to 110-fold differences in efficacy have been detected (Hodges et al., 1987). The sequences

28

M . J . Dtwro~ and R. C. HIDEg

of these variants are shown in Table 4 and they are listed in order of decreasing depolarizing activity. Invariant residues have been omitted. Some significant comparisons between depolarizing ability and structure are as follows: (i)

(ii)

(iii)

(iv)

(v)

(vi)

Naja naja siamensis CM 6 and Naja naja atra 1 (Fig. 19a). This pair of cytotoxins differs only in the transposition of residues 47/48 (AsnSer and SerAsn respectively) and yet the Naja naja atra toxin is about nine times less active. This transposition causes the weaker toxin to be the only representative without serine at position 48, and since this is not present in other toxin types, it must be virtually indispensable for activity. Naja nigricollis ~ and Naja mossambica mossambica I (Fig. 19b). Here, the substitution of Asn-59 for Asp in the latter toxin doubles the depolarizing ability, but it is not unique and has occurred in a further 8 cytotoxins. Furthermore, Asp is commonly found at this position throughout the ~-neurotoxin family, suggesting that it is more concerned with structure than with specific target discrimination. Naja naja siamensis CM7 and Naja naja naja I (Fig. 19c). With these toxins, the transposition of positions 50/51 and substitutions at 26 and 54 in the latter toxin reduce depolarizing ability 10-fold. The transposition also occurs in four other cytotoxins and replacement of Phe by Tyr at position 26 is very common. The change from Val to Glu is perhaps the most dramatic in chemical terms but it occurs in a further two cytotoxins and Glu is often present at this position in the short neurotoxins. Naja melanoleuca VII1 and Naja haje annulifera CM 2e (Fig. 19d). Seven substitutions are responsible for the large (19-fold) difference in depolarizing ability. All but one of these differences lie in the second and third loops. According to the sequence table, none of the substitutions is particularly unusual, so it is not possible to identify a critical change. The LDs0 value is increased in line with the depolarizing ability, but only by a factor of 3.5. Naja naja siamensis CM 7A and Naja naja siamensis CM 6 (Fig. 19e) The six residue differences between these variants produce a four-fold change in depolarizing ability. The locations of the substitutions are significant because they are located in, or adjoining, the segments where most of the evolutionary changes have taken place in the cytotoxin family. These are also very exposed areas of the molecular surface. Naja naja siamensis CM 7A and Naja mossambica mossambica IV (Fig. 19t"). This comparison is included to show how extensive changes can take place without alteration in depolarizing activity. It will be noticed that some of the changes have been associated with loss of efficacy in the other examples cited (e.g. Asp 59 to Asn 59, Ala 17 to Glu 17 and Phe 26 to Tyr 26). It is necessary to suppose, therefore, that potentially disadvantageous changes can be neutralized, or perhaps even made advantageous, by simultaneous changes elsewhere in the molecule.

Overall, Fig. 19 shows that the composition of the three major loops can have important consequences for depolarizing activity. In particular, it can be noted that the most marked differences in activity are associated with changes in the second and l~hird loops (i.e. Fig. 19a, e and f). While Fig. 19 is a limited selection of the available structure/depolarization data, the information contained in Table 4 can be simplified as a whole by excluding those residue positions whose pattern of variability does not reflect the trend of decreasing efficacy. For example, position 16 can accommodate glutamate, alanine or lysine, but the alternation of residue character does not correspond in any obvious way with the changing level of activity. Therefore, working on the assumption that each residue position has a fixed importance in the depolarization process, position 16 can be viewed as not being directly involved. Several positions can be excluded in this fashion, but some judgements are necessarily subjective. Those positions whose changing residue character does seem to have some relation to the degree of activity can be further simplified by highlighting

Elapid cytotoxins

29

Decrease x 2

c

=d) A-E

T--V

L-A

.........

~

~

..........

L-P

I~0

Fla. 19. Relationship between residue substitutions in cytotoxins and loss in depolarizing activity. Substitutions are displayed on a general backbone conformation. Comparisons are made between: (a) Naja naja siamensis CM6 and Naja naja atra I; (b) Naja nigricollis toxin ~ and Naja mossarnbiea rnossambica I; (c) Naja naja siamensis CM7 and Naja naja naja I; (d) Naja melanoleuea Vnl and Naja haja annulifera CM2e; (e) Naja naja siamensis CMTA and CM6; and (f) Naja naja siamensis CMTA and Naja mossambiea mossambica IV.

only the residue subsets that follow the toxicity trend. These residues are circled in Table 4 along with the substitutions for which there is only one example. The result of the simplification in the sequences represented in Table 4 is a general guide to the location and nature of the evolutionary changes that may have most influenced depolarizing ability. Considering only the circled residues in Table 4, it can be seen that some of the changes around the tip of the central loop are associated with the initial

30

M.J. DtwroN and R. C. HIDER

reductions in activity. For instance, substitution of serine-29 by alanine or lysine accompanies a loss in activity. However, when substitutions in this region co-occur with certain changes in the first major loop (e.g. Asn-4 to His-4), greater losses of activity result. Finally, when unusual residues appear in the third loop as well, quite catastrophic losses of activity are observed. In summary, therefore, it seems to be the residue variation in the three major loops (particularly at their extremities) which most influences depolarizing ability. Moreover, of the three loops, it is loop three where unusual substitutions have the most profound consequences. Although the problems of equating sequence with activity are partly to be expected, it is perhaps more surprising how some of the more general divisions amongst the cytotoxins fail to aid rationalization of the assay data. As described previously, the sequences can be readily marshalled into four combinatoric groups and at least two broad conformational groups on the basis of circular dichroism. However, these subgroups do not obviously correspond with differences in activity and neither do subgroups based on charge or species of origin. Cytotoxin efficacy has therefore to be pictured as being independent of some of the major manifestations of their structural and conformational evolution. 7.4.

STRUCTURE/ACTIVITY BY IMPLICATION FROM THE ~-NEUROTOXINS

Concentrating on major features only, there are two types of the related neurotoxins which are termed "long" and "short" according to the overall lengths of their polypeptide chains (Dufton and Hider, 1983). Both types show a high specificity for the nicotinic acetylcholine receptor, but they exhibit different toxicities in a wide range of animals. The short neurotoxins are generally more toxic to rodents and birds whereas the long neurotoxins tend to be more effective against reptiles and amphibians (Lim and Sawai, 1975; Burden et al., 1975). It has also been reported that 0c-bungarotoxin (a long neurotoxin) will bind tightly to the receptor from human skeletal muscle, but short neurotoxins will not (Ishikawa et al., 1985). Logically, given these two types of neurotoxin and the structures of the related cytotoxins, it should be possible to distinguish the residues that: (a) (b) (c) (d)

Confer specificity for the cholinergic receptor. Cause the difference in properties between the long and short neurotoxins. Confer specificity for the cytotoxin target. Maintain the characteristic molecular shape.

Much of this analysis had been previously discussed by Dufton and Hider (1983), but the outcome can be summarized thus: (i) The shape of the molecule depends mostly on the residues in the vicinity of the disulphide bridges. (ii) The prime targetting area of the neurotoxins comprises loop 3 and the adjacent part of loop 2 (i.e. structural residues apart, these are the areas of highest homology between the two types of neurotoxin). (iii) The directing of the neurotoxicity probably depends on the area involving loop 1 and the adjacent side of loop 2 (i.e. the major differences between the two types of neurotoxin are in this area). Also, loop 1 has been shown to be functionally important in the short neurotoxins (Harvey et al., 1984). (iv) Changes are required in all three major loops to convert a neurotoxin into a cytotoxin. A particularly significant feature is that the two most variable segments of the cytotoxins lie very close to the sites where the major differences (which include residue insertions and deletions) exist between the short and long neurotoxins (Fig. 20). If a parallel is to be drawn, therefore, the highly conserved area of the cytotoxins involving loop 3 and the adjacent strand of loop 2 can be regarded as the fundamental targetting region with the

Elapid cytotoxins

31

variable sections of loop 1 and 2 acting as supplementary directing influences. Such an extrapolation also carries with it the need for a type of target which is similar to the nicotinic cholinoceptor. Reviewing the assay data in the light of this hypothesis, it is consistent in the sense that evolutionary changes in all three molecular environs of the cytotoxins (i.e. the shapedetermining, conserved recognition and variable recognition areas) can cause a change in overall efficacy. As implied by the permitted evolutionary variation, alteration to the conserved recognition site (Fig. 21) could be expected to have a greater effect on toxicity than alteration to the variable recognition sites. While this generalization probably requires qualification according to the chemical "severity" of any given change, it will be recalled that the depolarization data do support the contention that loops 2 and 3 are particularly important (Table 4). Regarding individual residue positions, there are only 3 residues in the neurotoxins which can be considered as absolutely essential for targetting, and these are Trp 29 and ArgGly 33/34 (corresponding to cytotoxin positions 27 and 31/32 respectively) (Dufton and Hider, 1983). In the cytotoxins, residue 27 is very highly conserved as Met, This is a significant evolutionary interchange because Trp and Met are the two least common amino acids (each has only one codon in the genetic code), so one evolutionary "rarity" has been replaced by another. Furthermore, chemical modification of both Met 25 and Met 27 is known to increase LDs0 (Carlsson and Louw, 1978) and to decrease depolarizing abilities in some cytotoxins (Wong et al., 1980; Hodges et al., 1987). The ArgGly dipeptide unit corresponds to positions 31 and 32 in the cytotoxins. These positions are part of the variable segment at the trip of the central loop and, if the analogy with the neurotoxins is pursued by supposing that they are functional focal points, it follows that there may be more than one version of the cytotoxin target. The structure/activity data for the cytotoxins does show that changes in this area might influence efficacy (Table 4), and since the sequence data reveals there to be basically two versions of the cytotoxin 29-32 segment, at least two, if not more, related targets are a possibility. A fourth residue which is known to

]

|

it~ s

s

os j t

s s--.d

s l

++

x

~P

\,"

s -o--I sI

FIG. 20. (a)The superimposed a-carbon skeletons of a-cobratoxin (Walkinshawet al., 1980) and erabutoxin b (Low, 1979). The segments shown in dashed lines highlight the major structural differencesbetweenthese two typesof neurotoxin.(b) The predicteda-carbon skeletonof a typical eytotoxin. Those areas of the chain which show the greatest variation in residue character are shown as a dashed line. It can be noted that these areas of variation correspond to those highlighted in (a).

32

M.J. DuFToN and R. C. HmEg

FIG. 21. Possible cytotoxin interaction site. The backbone structure of a typical cytotoxin is presented, the three extended loops being numbered 1, 2 and 3. Conserved hydrophobic residues are denoted by the shaded circles.

be very important functionally in the neurotoxins is a lysine which is located in a position analogous to the conserved serine 48 of the cytotoxins. As shown in Fig. 19a and Table 4, loss of this serine can be associated with major losses of depolarizing activity. The neurotoxins are not the only molecules to show specificity for the cholinoceptor and this has prompted searches for the common structural requirements. In this context, it has been noted that some of the conserved targetting residues in the neurotoxins mimic the structure of curare and related alkaloids by way of their chemical characteristics and juxtapositions (Dufton and Hider, 1977 and 1980). Should the reactive site of the cytotoxin molecule prove to encompass positions 27, 31, 32 and 48, then in a similar fashion to the neurotoxins, it is reasonable to suppose that the cytotoxin target is also exploitable by other types of molecule and that a low molecular weight mimic of a cytotoxin may either already exist, as for instance an alkaloid, or be achieveable synthetically. Such molecules might possess muscle depolarizing activity and would be of considerable pharmacological and clinical interest. 7.5. CONCLUSIONS ON STRUCTURE/ACTIVITY

The general picture that emerges of cytotoxin structure/activity is one in which the three large and exposed polypeptide loops feature heavily. On the basis of the evolutionary conservation of residues and the effects of substitutions on relative efficacy, the area in which loops 2 and 3 adjoin each other is especially prominent (Fig. 21) and is expected to provide the prime targetting influence. In terms of conformation too, it is clear that as a result of the triple stranded fl-sheet in the molecule, this area is also the most conformationally defined part of the emergent loop structure. The chain segments peripheral to this targetting focal point cannot be dismissed as superfluous, however. As the assay and spectroscopic evidence has indicated, some changes in efficacy and conformation accrue from residue changes in these segments, so it is prudent to assign the latter a role in modulating the central toxic theme of these molecules.

Elapid cytotoxins

33

8. CONCLUDING DISCUSSION The preceding sections have shown that the greatest area of doubt surrounding the cytotoxins is the exact identity of their cell membrane target. The following summarizes the implications of the different approaches to the problem and then explores some of the reasons why the target should prove so elusive. Finally, attention is given to an aspect of the cytotoxin story which seldom receives satisfactory explanation, namely the relevance of the partnership with phospholipase Av 8.1. IMPLICATIONS OF SEQUENCE COMPOSITION

At the simplest level, the amino acid compositions of the cytotoxins indicate that their site of action has a complementary environment in which hydrophobic and negatively charged moieties feature. In a typical animal cell membrane, such an environment could be provided by negatively charged lipids, membrane proteins, or both. Such lipids are important membrane components, but as pointed out in the earlier discussion (Section 6.2) they are predominantly disposed on the cytoplasmic faces of membranes. Membrane proteins, in contrast, often possess clusters of negatively charged carbohydrate and amino acid residues at the extracellular boundary of their bilayer spanning segments. Therefore, in terms of accessibility from the extracellular phase, membrane proteins are more likely to provide the necessary binding environment. 8.2. IMPLICATIONSOF STRUCTURE It is virtually certain that the cytotoxins and the neurotoxins adopt very similar tertiary structures and conformational balances. The inescapable inference, therefore, is that the prime binding site for cytotoxins is related to the binding site exploited by ~-neurotoxins in their action on the nicotinic cholinoceptor. Admittedly, without a knowledge of the neurotoxins, this notion could not be entertained on the basis of the remaining evidence. However, it is difficult to envisage any other reason for the retention of such a characteristic molecular shape in both toxin groups. In addition, the evolutionary trends within both groups are similar, so whatever the nature of the cytotoxin target site, it is "guiding" cytotoxin evolution in much the same way as the nicotinic receptor site guides neurotoxin evolution. Using the nicotinic cholinoceptor as a model, the cytotoxin target site is implied as involving an oligomeric transmembrane protein which governs ion fluxes. Indeed it is rapidly emerging that there is a common structural design for cell membrane channels (Unwin, 1986). The studies on muscle preparations suggest that an endogenous calcium channel is the most probable acceptor. 8.3. IMPLICATIONS OF ASSAYS The apparent consensus of the various pharmacological assays is that when a cell encounters a cytotoxin, its integrity is damaged in a manner which both renders its membrane susceptible to phospholipase A2 attack and disturbs its normal ion fluxes. From what is known about the melittin/phospholipase A2 interplay in bee venom, the synergism between the cytotoxins and phospholipase A2 may show that binding brings about aggregation of membrane proteins. Normally, membrane proteinS (together with their attached carbohydrate moieties) form an effective physical protection for the lipid bilayer when they are uniformly distributed across the cell surface. Thus, in the absence of any aggregating influence, phospholipase A 2 may not be permitted direct access to the most vulnerable parts of the lipid bilayer. The aggregation could either be caused by the cytoroxins directly cross-linking the membrane proteins upon binding or causing a depolarizationdependent contracture of the cell membrane cytoskeleton. By the same token the depolarization effects could be caused by the cytotoxins binding directly to the ion-flux regulators JF]"36/1--C

34

M . J . DUFTON and R. C. HIDER

or by influencing the latter by way of changes triggered in the pervading cytoskeleton. In both scenarios, the major outcome of cytotoxin binding is a physical change over large areas of the cell surface mediated by the membrane cytoskeleton, so a wide variety of membrane mechanisms could ultimately be influenced. This concept of cytotoxin action, particularly the hypothesized aggregating ability, provides an interesting link with some of the more curious proposals about cytotoxin evolution. In 1980, Drenth et al. noted that there was a similarity between the fundamental folding pattern of the 0t-neurotoxins (and thereby, the cytotoxins) and wheat-germ agglutinin, a plant lectin. Like the cytotoxins, there has been some difficulty in establishing a specific target for the lectins, but it is thought that the wheat-germ agglutinin is selective for saccharide-containing receptors on the cell surface. While it is the ability to bind saccharide that seems to be at the forefront of lectin action, cross-linking of glycoproteins or glycolipids in the membrane has been proposed as one of the major consequences of the binding (Barondes, 1981). Drenth et al., (1980) and Strydom (1977) have also noted a similarity between the disulphide bridge arrangement of the toxins and that of ragweed pollen allergen. In common with other allergens, this protein causes the release of histamine from cells by aggregating immunoglobulin E receptors present in their membranes. 8.4. WHY IS THE TARGET OBSCURE? Given these pointers to the nature of the cytotoxin target, why has it proved difficult to identify? Again, possible explanations are also forthcoming from the known data. (1)

(2)

(3)

According to structural analyses of the nicotinic cholinoceptor in different animals (Popot and Changeux, 1984) and the incidence of neurotoxins throughout the elapid snakes, this receptor is both fundamental and largely invariant in a very wide range of living organisms. However, in comparison with the neurotoxins, cytotoxins have a rather restricted incidence (i.e. only the cobra species Naja and Hemachatus) so this may mean that their target is a development peculiar to the prey taken by these particular species. Since, in terms of the biochemical regime of an organism, a recent evolutionary development may be more peripheral and more limited in its application, a typical membrane may contain relatively small amounts of the target molecule. Consequently, selection of suitable assay material becomes crucial, especially if the target is a higher order control mechanism which can trigger a variety of measurable effects indirectly. The other possibility which must be entertained in this context is that the cytotoxin target may indeed be as widely distributed as the nicotinic receptor, but its exploitation as a toxin target has not been implemented by many elapid snakes through lack of evolutionary opportunity or necessity. The neurotoxin/postsynaptic membrane interaction may be giving a distorted impression of the general mechanistic theme that underlies both neurotoxins and cytotoxins. This is because the postsynaptic membrane is unrepresentative of a typical membrane. It is largely composed of densely packed receptor molecules and so the content of lipids and other membrane proteins, and their relative degrees of physical freedom, is unusual. The potential for such effects as membrane protein aggregation is therefore minimal, even though the neurotoxins might be imposing the tendency by binding to two subunits (Hucho, 1979; Muhn et al., 1984). Moreover, the "fine tuning" of the postsynaptic membrane as a whole, which enables very fast response/recovery, could result in parts of the basic toxic mechanism being emphasized or minimized to an extent sufficient to remove obvious similarity to the cytotoxins. The consequence of this is that a more complete understanding of cytotoxin action may cast new light on details of neurotoxin action which have hitherto not been realized or measured. As shown in Table 1, the cytotoxins are divisible into subgroups on the basis of sequence. Coupled with the large number of cytotoxin variants that can be found in

Elapid cytotoxins

35

the venom of a single species, this might indicate the existence of several distinct versions of the cytotoxin target. It is known for some enzymes (e.g. lactate dehydrogenase) that certain tissues often have specialist versions of the protein, therefore some cytotoxins may differ from each other in terms of tissue specificity within the same prey animal. This could in part explain why LDs0, hemolysis and depolarization data are not complementary in their ranking of cytotoxin variants. If the cytotoxin target is an oligomer of homologous protein subunits, different versions could be obtained by permutating subunit composition and arrangement. Indeed, this possibility has been put forward to account for aspects of cytotoxin evolution (Breckenridge and Dufton, 1987).

8.5. THE ROLE OF PHOSPHOLIPASEA 2 The ability of cytotoxins to synergize with phospholipase A2 is an important property that appears to set them apart from the mode of action of the ~-neurotoxins, but there are parallels to be drawn in this respect too. Many of the venoms which contain a-neurotoxins also contain phospholipase A2 homologues which are specific for presynaptic membranes. These enzyme homologues have been termed fl-neurotoxins and they block the release of acetylcholine and otherwise damage the membrane. Since an individual neurone is in fact a single specialized cell, it can contain both acetylcholine receptors and acetylcholine release mechanisms. In theory, therefore, sites for both types of neurotoxin could be present in the same membrane. In practice, however, the ~t-and fl-neurotoxins are specific for the neuromuscular junction where the two types of target are in separate membranes. Nevertheless, given that a neuronal cholinergic receptor is probably very similar to that of the neuromuscular postsynapse (i.e. it can bind x-bungarotoxin, a homologue of a-bungarotoxin) (Grant and Chiappinelli, 1985), it is still appropriate to regard the two fundamental types of neurotoxin as complementing each other's action on the same cell. In cells not specialized for electrical/chemical transmission, the mechanisms that control release and reception of chemical transmitters are not localized to such extremes and are more evenly distributed. For these cells, toxins and enzymes based on the neurotoxins could bind in each other's vicinity, so this can be contemplated as applying to the cytotoxin/phospholipase A2 system. 9. CONCLUSION Seldom in the study of a protein family is there such a disparity between what is known about the structure and evolution of a protein and what is known about its site of action. Some investigators, such as the present authors, rely heavily on the detailed conformational data and evolutionary relationships in attempting to deduce the nature of the cytotoxin target. Others, however, start from the undoubted effects that these toxins have on a variety of lipid and tissue systems and do not prejudice the issue by expecting any similarity to the mode of action of the ~-neurotoxins. This has led, at the present time, to two very different expectations: on the one hand, that the cytotoxin target is a membrane protein, and on the other hand, that membrane lipids and the bilayer itself constitute the binding site. Both approaches have one particular weakness that can be singled out. For the structure-based approach, much depends on the current concept of ~-neurotoxin action, but there is still much to be learned about this mechanism. As already pointed out, the postsynaptic membrane is very specialized and so properties of the cytotoxins which are in fact shared by the neurotoxins could be masked or exaggerated. For the purely experimental approaches on lipid systems, phospholipase A z contamination is a perennial problem. Assays for the enzyme may prove negative, but it is worth remembering that some of the neurotoxic phospholipase A2 homologues in elapid venoms have no detectable enzymic activity. This does not mean to say, of course, that their bilayer penetrating ability, for example, has also been impaired.

36

M.J. DUFTONand R. C. HIDER

If the two views of cytotoxin action are to be finally clarified, the acceptor molecule must be unambiguously identified and isolated. According to the evidence so far obtained, future attention should be directed at the properties of membrane cytoskeletons and their behavior in association with the lipid bilayer and transmembrane ion movements. Acknowledgements--We are grateful to Professor H. Rochat, Dr. P. Bougis and Mr. I. Morrison in respect of certain diagrams used in this review. We would also like to acknowledge the assistance of Professor A. L. Harvey as regards the pharmacological data, and Margaret Smith for her careful and patient word-processing.

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