J . TOXIC0L.-TOXIN REVIEWS, 17(2). 213-238 (1998)

STRUCTURE, FUNCTION AND BIOPHYSICAL ASPECTS OF THE MYOTOXINS FROM SNAKE VENOMS

Charlotte L. Ownby

Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A.

ABSTRACT

Snake venom myotoxins can be categorized into three types: small, basic polypeptides

such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A, toxins

such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction

of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide

myotoxins is different from that induced by the cardiotoxins and phospholipase Az myotoxins in

that the former d o not appear to lyse the sarcolemma whereas the latter two types cause lysis of

the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they

are all highly basic proteins, and a large portion of their surface charge is positive Also, they all

have considerable P-sheet structure which may be involved in interaction with the membrane The

purpose of this review is to describe the structure and function of these myotoxins and to evaluate

features they might share which could shed light on their mechanism of myotoxic action

INTRODUCTION

Myotoxins from snake venoms can be classified according to their molecular size, the

presence or absence of catalytic activity and the nature of their action on muscle cells Since the

term “myotoxin” has been defined differently by different authors (l-4), it is perhaps wise to

213

Copyright 0 1998 by Marcel Dekker, Inc

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define “myotoxin”as it will be used in this review. Here, myotoxin is defined as a protein

produced by snakes in their venom glands, which through direct chemical action injures or kills

mammalian skeletal muscle cells. This definition is similar to that of Hams (3) but varies from the

definition of Gopalakrishnakone et al. (4) in which a distinction is made between “general

myotoxins (myoglobinuric myotoxins)” and “local myotoxins (myonecrotic toxins)”. General

myotoxin is defined as a toxin which causes “systemic myonecrosis”, i.e., causes necrosis of

muscle cells which are located away from the site of injection. According to the definition,

“general myotoxins” induce myoglobinuria and have higher LD,, values, whereas “local

myotoxins”do not induce myoglobinuria and have lower LD,, values. However, several

myotoxins classified as “general myotoxins” under this scheme (4) have low LD,, values, i.e.,

crotoxin, mojave toxin, notexin and taipoxin whereas some other toxins classified as “local

myotoxins” (several B. usper and V. rzrsselli myotoxins) have rather high LD,,values. For many

myotoxins, the presence of myoglobinuria has not been noted, nor have LD, values been

determined. Thus, the term “myotoxin” will be used in this review to refer to any proteinaceous

substance in the venom of snakes which induces injury, death or impairs the function of skeletal

muscle cells, and no distinction will be made based on the production of myoglobinuria.

Myotoxins in snake venoms can be classified into three main types: (1) low molecular

weight, non-catalytic peptides such as myotoxin CI and crotamine; (2) cardiotoxins from cobra

venoms; and (3) proteins with phospholipase A, structure. Hemorrhagic metalloproteinases from

some crotaline venoms induce myonecrosis, but there is no convincing evidence yet that these

toxins act directly on the muscle cell, and they will not be addressed in this review.

Myotoxins have been isolated from the venoms of snakes from the families Elapidae,

Hydrophiidae and Viperidae, but not Colubridae. The family Elapidae includes the cobras, kraits,

mambas and coral snakes, and venoms from these snakes contain phospholipase A,-type

myotoxins as well as cardiotoxins. Members of the Hydrophiidae family, the sea snakes, contain

phospholipase A,- type myotoxins in their venoms. One subfamily ofthe family Viperidae, the

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MYOTOXINS FROM SNAKE VENOMS 215

viperinae (true vipers), contains members which have phospholipase A,-type myotoxins, while

members of the other subfamily, the crotalinae (pit vipers), have venom which contains

phospholipase A,-type myotoxins as well as the low molecular weight, non-enzymatic polypeptide

myotoxins.

SMALL, BASIC POLYPEPTIDE MYOTOXINS

Several small, highly basic, non-catalytic myotoxins have been isolated from venoms of

snakes in the family Viperidae, subfamily Crotalinae, genus Crotnlns. The first of these to be

isolated was crotamine, from the venom of t h e tropical rattlesnake, Crotahs dnrissns /err/pcirs

(5). Bober et al. (6) using immunodiffusion and ELISA, detected similar proteins in venoms from

snakes in the Crotnliis and Sis/rririrs genera, but not in venoms of snakes in six other snake genera

(Agkistrodon, Bitis, Bothrops, Cnlloselnsnin, Nnjn or Jfipern).

Structure and Biophysical Aspects. Since these toxins are highly basic, pI>9.0, they can be

easily isolated from the crude venom by cation exchange chromatography using Sephadex C-25 or

a similar medium (7). The amount of these toxins in crude venoms varies, but Goncalves (8)

found that the percentage of crotamine per venom ranged from 9.6 mg% to 53.7 mgYo in 25

venom samples analyzed. Crotamine molecules tend to form aggregates at high pH values of 12.5

(9).

The amino acid sequences for six of these toxins are shown in Figure 1. In general, they

contain about 42 amino acids with three disulfide bonds. Griffin and Aird (10) suggested a new

nomenclature for these toxins, i.e., named by length instead of by trivial name related to snake

venom of origin. The C-terminal is the least conserved portion of the molecule whereas the N-

terminal is always a tyrosine (1 1).

Also, it appears that multiple forms of the toxin could be present in the same venom. Aird

et al. (12) and Griffin and Aird (10) reported multiple forms of myotoxin n from the venom of one

specimen of Crotnhts viridis viridis. Recently, Nedelkov and Bieber (1 3) demonstrated the

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Crotamine" Myotoxin a" viridis 2'' viridis 3" viridis 4" Peptide c'' cvc I" cvc 11'' CVC IImicro'' CAM20

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG YKQCHKKGGHCFPKEKICIPPSSDLGKMDCRWKWKCCKKGSG Y K R C H K K E G H C F P K T V I C L P P S S D F G K M D C R W K C C K K G YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSWA YKRCHKKEGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSVN YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSVN YKRCHKKEGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSVN YKRCHKKGGHCFPKEKICTPPSSDFGKMDCRWKWKCCKKGSVN YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWRWKCCKKGSVN YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWRWKCCKKGSV

Superscript indicates reference from which sequence was taken

Figure 1. Primary Sequences of Selected Myotoxic Polypeptides

existence of many isoforms in several rattlesnake venoms using capillary electrophoresis and

matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, cloning and

sequencing results from the sequencing of four genes for crotamine (14) predicted the presence of

several variants of crotamine in Crotnlus ditrissm terrijhs venom which were confirmed by

sequencing purified crotamine and its CNBr fragments. Variations were found in residues in

positions 3 (arginine for glutamine), 6 (isoleucine for lysine), 15 (glycine for glutamic acid), 19

(isoleucine for leucine), 3 1 (proline for arginine), and 34 (arginine for tryptophan). The gene for

myotoxin a has also been cloned and sequenced (1 5, 16). and the gene encodes a 22-amino acid

signal peptide, and a 43-residue polypeptide, the sequence of which corresponds to the published

sequence for native myotoxin a, and an additional lysine on the COOH-terminal end. Apparently

both crotamine and myotoxin a have a terminal lysine which is removed during post-translational

processing.

The secondary structure for myotoxin a was determined, using three theoretical

approaches (21), to contain significant amounts of P-sheet and p-turn structures, but only a small

amount of random coil and a-helix. Similar results were obtained with crotamine (22), and

Beltran et al. (9) showed that crotamine was a flattened or elongated molecule, not spherical.

Later, Henderson et al. (23) found, using both one- and two-dimensional NMR spectroscopy and

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MYOTOXINS FROM SNAKE VENOMS 217

the pH dependence of chemical shifts, that the amino terminal region of myotoxin a forms a helix

which puts Tyr-1, His-5 and His-10 in close proximity to each other. To date the crystal structure

of these toxins has not been solved.

Function. Much of the early work on the biological activities of these small myotoxins has been

previously reviewed (1,2). Their action is characterized by the instantaneous induction of muscle

contracture upon intramuscular injection into mice, followed by some degree of respiratory

distress. They have also been described as inducing extreme tonic paralysis of the hind limbs of

mice, but the question of their possible neurotoxic activity has not been adequately addressed. If

the dose of toxin is sublethal, the mice will experience some distress for 15-30 minutes, then

recover without any apparent injury. If the dose is lethal, the mice usually die within 5- IS

minutes after injection. The induction of hypotension and shock has also been described for a

peptide from the venom of Crotnlrrs vindis helleri (24), and these pathophysiological responses

could lead to death.

Effects on Muscle Cells In V i i u The pathogenesis of myonecrosis induced by myotoxin

a has been documented by Ownby et al (25) using light and electron microscopy. Myotoxin a

and homologous proteins induce myonecrosis of skeletal muscle cells which is of a relatively slow

onset, beginning with dilatation of the sarcoplasmic reticulum as early as 30 minutes after

injection of the toxin. Examination of tissue with the light microscope at 3- 24 hours after

injection of toxin reveals the presence of vacuoles in affected muscle cells The initial

morphological change appears to be dilatation of the sarcoplasmic reticulum and the perinuclear

space. The typically flattened cisterns of sarcoplasmic reticulum become rounded and enlarged,

and they increase in size as the degeneration proceeds. At about 24 hours after injection,

depending on the dose used, the sarcoplasmic reticulum is grossly enlarged, disorganized and

occupies much of the cytoplasm. By 48 hours after injection, the sarcoplasmic reticulum has

broken down into numerous smaller vesicles, and at this time there appears to be some myofibril

breakdown. Also, by this time mitochondria are swollen and lack some cristae, but they are not

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completely degenerated. However, at this time period, the T-tubules are still normal in their

appearance. Intact sarcomeres are separated by loose actin and myosin filaments, and it appears

that dissolution of myofilaments occurs first between Z lines. By 72 hours, most affected muscle

cells contain completely disorganized myofilaments as well as swollen and damaged mitochondria.

It is interesting that even in cells with extensive damage, the sarcolemma and basal lamina remain

morphologically intact. There are no pathologic changes in other cells such as macrophages,

fibroblasts or the endothelial cells of capillaries. It appears that these toxins are specific for

skeletal muscle cells. Ownby et al. (25) interpreted these results as suggesting that the toxin acted

on the plasma membrane of the skeletal muscle cell to alter the transport of sodium ions, leading

to a net influx of Na' into the cell. Water follows down its concentration gradient, the

sarcoplasmic reticulum takes up the water, and becomes swollen. At some point, the

sarcoplasmic reticulum cannot take up any more water. The inability of the cell to regain its

osmotic equilibrium leads to hrther pathological changes in the cell such as the breakdown of

myofilarnents. Crotamine induces essentially the same type of pathogenesis of myonecrosis (26,

27) as do two similar myotoxins from the venom of Crofnlm viridis concolor (28).

Effects on Cells I n Vitro Studies on cells in culture have attempted to delineate the

actual mechanism of myonecrosis Myotoxin I from Crotnhrs viridis coricolor venom caused a

marked increase in the percentage of primary rat myoblasts which undergo contraction (29).

Later Ziolkowski et al. (30) showed that myotoxin n has the same effect, and that myotoxin a

induces contraction in myotubes from primary rat myoblasts which stopped contracting after

placement in serum-free defined medium. The toxin induced contractions within 2-3 minutes after

application, but it took one day for the percentage of contracting myotubes to reach a maximum

level. These authors concluded that their data are consistent with activation of the voltage-

sensitive sodium channel by these toxins.

Baker et al. (31) found that myotoxin n bound to L8 cells, a rat skeletal muscle-derived

cell line, in a saturable, reversible manner with a moderate binding affinity. The binding site was

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MYOTOXINS FROM SNAKE VENOMS 219

not accessible to pronase even when the experiments were done at 4°C. indicating that even at

low temperatures, the myotoxin is either inserted into the membrane or bound to a receptor

partially obscured from the surface of the cell They did not find the same induction of

contraction in the L8 cells which Ziolkowski et al (30) found in primary rat myoblasts, nor did

they observe any pathologic effects on these cells However, at doses higher than those producing

binding saturation, the toxin did inhibit cell growth in a dose-related manner.

Mechnnisni of Toxin-Induced Muscle Cell Dnmnge Although the precise mechanism

by which these toxins damage skeletal muscle cells is still not known, there have been a couple of

lines of investigation which have shed some light on how these molecules interact with skeletal

muscle cells.

One line of investigation has focused on the interaction of these toxins with the Ca2+ -

ATPase of the sarcoplasmic reticulum ( 3 2 - 3 5 ) . Tu and Morita (32) reported that peroxidase-

conjugated myotoxin a attached to the sarcoplasmic reticulum of skeletal muscle from human

biopsies. These investigators interpreted these data as indicative that the primary target of

myotoxin a is the sarcoplasmic reticulum However, since the methodology in this paper involved

appIication of the peroxidase-conjugated toxin to muscle tissue which had been sectioned, it is not

clear whether the toxin actually reaches and binds to the sarcoplasmic reticulum of intact skeletal

muscle cells when the toxin is applied in viva However, later Volpe et al ( 3 3 ) showed that

myotoxin a inhibited calcium loading into isolated SR vesicles in a dose, time and temperature

dependent manner and stimulated CaZ' -dependent ATPase This study indicates the ability of

myotoxin a to interact with the Ca2' - ATPase Based on these studies, the authors concluded that

myotoxin a attaches to the CaZ'-ATPase of the SR and uncouples Ca2* uptake from Ca"-

dependent ATP For this mechanism to be part of the I H vivo action of myotoxin a, the toxin

would have to be internalized by the cells, and so far there is no evidence that this occurs

Utaisincharoen et al. (35) have shown using isolated SR vesicles and "'I-myotoxin n that the

toxin can be cross-linked to two SR proteins The protein in the SR to which myotoxin a bound

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was thought to be a 53-kDa glycoprotein which serves as a modulator of the SR Ca2'-ATPase.

As pointed out by these investigators, this work was done using isolated SR, and does not

constitute evidence that myotoxin a binds to these SR proteins in viva.

The important question which remains unanswered is whether myotoxin n actually enters

the skeletal muscle or interacts only at the extracellular surface of the plasma membrane. Studies

ofL8 muscle-derived cells in culture ( 3 1) strongly suggest that myotoxin a binds to the plasma

membrane and is either inserted into the membrane or bound to a receptor which is partially

obscured from the surface of the cell.

A second line of investigation has tried to address the question of how these toxins

interact with the plasma membrane of the muscle cell. Many of these studies indicate that these

toxins alter the hnction of voltage-sensitive sodium channels of the skeletal muscle cell plasma

membrane, increasing the influx of Na' leading to depolarization and contraction. Studies using

both crotamine (36) and myotoxin a (37) have shown that these toxins increase the resting

membrane permeability to sodium, probably by altering the sodium channel of the sarcolemma at

a site which is distinct from that oftetrodotoxin, veratridine or sea anemone I1 toxin. This

interaction could lead to an increase in intracellular Na' concentration followed by an increased

influx of water leading to swelling of the sarcoplasmic reticulum as this organelle takes up water

from the cytosol. If the binding to the Na' channel is irreversible, the permanent alteration in

osmotic balance could lead to cell swelling and necrosis.

Only two studies have investigated the effects of these toxins on lipid membranes. Liddle

et al. (38) looked at the interaction of myotoxin a with model lipid membranes using calorimetric

techniques whereas DuFourco et al. (39) used changes in intrinsic fluorescence to detect changes

in the same system. However, both of these studies support evidence that myotoxin a interacts

with the plasma membrane of the muscle cell, but neither study could define the precise chemistry

of the interaction.

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MYOTOXINS FROM SNAKE VENOMS 22 1

Structure-Function Relationship. It is interesting to ask the question of how the structure of

these small peptides relates to their myotoxic activity. Kini and Iwanaga (40) pointed out that all

ofthese myotoxins have a cationic region at residues 2-10 which is followed by a hydrophobic

region, and they proposed that this region might be important in myotoxicity.

Mori et al. (34) produced a “nicked” myotoxin u by cyanogen bromide cleavage of the

native molecule and found that the “nicked toxin had a conformation similar to that of the

original toxin, was myotoxic and inhibited calcium ion loading activity. Apparently, breaking the

Met(28)-Asp(29) bond did not destroy the biological activity of the toxin even though it did cause

a slight increase in the random coil nature of the molecule.

Henderson et al. (23) looked at myotoxin a with NMR spectroscopy. They found using

both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts that

the amino terminal region of myotoxin a forms a helix which puts Tyr-1, His-5 and His10 in close

proximity to each other Other studies found that chemical modification of tyrosines and

histidines reduced the contraction-inducing ability of the toxin (30) on primary rat myoblasts in

culture. Thus, the N-terminal hydrophobic-cationic region might be important for interaction with

the membrane, but it is not known whether this interaction is through phospholipids or specific

proteins in the membrane.

CARDIOTOXINS

Cardiotoxins are proteins with primary action on the heart found in venoms from snakes

belonging to two genera in the Elapidae family (cobras, kraits, mambas and Australian tiger

snakes), i.e., Naju (the cobras) and Heniachati/s (the Ringhals) However, since they appear to

lyse a wide variety of cells, they have also been called direct lytic factor, cytotoxin, membrane-

active polypeptide and membrane-disruptive toxin (41 ) These toxins also induce depolarization of

skeletal muscle cells and can lead to necrosis. There are several excellent reviews of cardiotoxin

structure and function (41-43) As most authors point out, there is one major problem in

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working with the cardiotoxins. It is difficult to isolate them from crude venom without CO-

isolation of a phospholipase A,. Hodges et al. (44) indicate that even procedures such as gel

chromatography in the presence of 8 M urea, hydrophobic chromatography and immunoaffinity

chromatography may not lead to a cardiotoxin preparation completely free of phospholipase A,.

Structure and Biophysical Aspects.

Primary Structure. Cardiotoxins are polypeptides containing 60-63 amino acid residues

crosslinked by four disulfide bonds. Over 50 variants have been isolated, characterized and

sequenced (45). Dufion and Hider (42) classified them into five subgroups as follows: (1)

Subgroup A - contain ProlO and Pro3 1; (2) Suberouo B - does not contain Pro1 0, has Pro3 1; (3)

Subgrouo C - contain ProlO and not Pro3 1; and (4) SuberouD D- does not contain either ProlO

or Pro3 1. (5) Suberouo E - cytofoxin homologous - classification based on the polypeptide chain

length, some have an extra residue between the first two half-cystines, and the possession of

distinct triresidue sequences between the fourth and fiAh half-cystines (AAT, ADA, TDT, and

TDA). Subgroups A-D are more similar to each other than to proteins in Subgroup E, and the

latter vary mostly in residues at positions 10, 11 and 12 as well as at positions 29 to 32, with the

best grouping obtained using presence or absence of Pro at positions 10 and 3 1. To illustrate

these groupings and to show the primary structure of cardiotoxins, selected sequences are shown

in Figure 2.

More recently, Chien et al. (46) classified cardiotoxins into two types, S- and P-type,

based on their interaction with phospholipids. Even though all cardiotoxins have a phospholipid

binding site in loop 1, P-type cardiotoxins are characterized by the presence of Pro-3 1 and

another phospholipid binding site near the tip of loop 2 whereas S-type cardiotoxins have Ser at

position 29 and a loop 2 which is more hydrophilic. P-type cardiotoxins have higher fusion and

binding activities than the S-type molecules which may be due to the possession of an additional

phospholipid binding site in loop 2 of the P-type molecules.

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MYOTOXINS FROM SNAKE VENOMS 223

Subgroup A 1.LKCHQLVPPFWKTCPEGKNLCYKMYMVATPMIPVKRGCIDVCPKNSALVKYMCCNTDKCN ( S ) 2.LKCHKLVPPFWKTCPEGKNLCYKMYMVATPMIPVKRGCIDVCPKNSALVKYMCCNTNKCN ( P I 3.LKCNRLIPPFWKTCPEGKNLCYKMTMRLAPKVPVKRGCIDVCPKSSLLIKYMCCNTNKCN ( P ) 4.LKCNQLIPPFWKTCPKGKNLCYKMTMRAAPMVeVKRGCIDVCPKSSLLIKYMCCNTDKCN ( P )

Subgroup B 5.LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN ( P ) 6.LKCNQLIPPFWKTCPKGKNLCYKMTMR~P~PVKRGCIDVCPKSSLLIKYMCCNTNKCN ( P )

Subgroup C 7.LKCHQLVPPFWKTCPEGKNLCYKMY~SSSTVPVKRGCIDVCPKNSALVKYVCCNTDKCN ( S ) 8.LKCYKLVPPFWKTCPEGKNLCYKMY~STLTVPVKRGCIDVCPKNSALVKYVCCNTDKCN ( S )

Subgroup D 9.LKCNKLIPIASKTCPAGKNLCYKMFMMSDLTIPVKRGCIDVCPKSNLLVKYVCCNTDRCN ( S )

10.LKCNKLIPLAYKTCPAGKNLCYKMEVSNKTVPVKRGCIDACPKNSLLVKYVCCNTDRCN ( S ) 11.LKCNKLIPIAYKTCPEGKNLCYKMMLASKKMVPVKRGCINVCPKNSALVKYVCCSTDRCN ( S )

Subgroup E 12.IKCHNTLLPFIYKTCPEGQNLCFKGTLKF-PKKTTYNRGCAATCPKSSLLVKYVCCNTNKCN ( P ) 13.LKCHNTQLPFIYKTCPEGKNLCFKTTLKKLPLKIPIKRGCAATCPKSSALLKSVCCNTDKCN ( P ) 14.LICHNRPLPFLHKTCPEGQNICYICMYLKKTPMKLSVKRGCAATCPSERPLVQVECCKTDKCNW 15.LKCHNKLVPYLSKTCPDGKNLCYICMSMEVTPM-IPIKRGCTDTCPKSSLLVKWCCKTDKCN

(1)Naja haje hoje: CM-7; (2)N hnje onniilifero:CM-6; (3 )Nm mossombrco:Vn3; (4)N.nrgricollis: Cardiotoxin; (5)N.noja otra: cardiotosin; (6 ) N m.mossombicn: Vnl ; (7) N.h. hoje: CM-8, (8) N. haje annulifera: CM-8; (9) N.n.ntra: 1; (10) N.n. siornensis (Kaouthia): CM-7; ( I I ) N.m.mossombica:V" 4; (12) N. rnelanoleuca: Vn 2; ( 1 3 ) N hqje onniiiflero: CM-13A: (14) Hemochatiis hemochnriis: 9B; (15) Hemachatus hemochatiis: 1 IA. Note: (S) = S-type: (P) = P-hpe, according to Chien ct al (46).

Figure 2. Primary Sequences of Selected Cardiotoxins (Adapted from reference 42)

Secondary and Teriiary Structure. The structure of several cardiotoxins has been solved

by X-ray crystallography, and more recently, by NMX (for review see 42). X-ray crystallography

data indicate that the three-dimensional structure of cardiotoxins is distinguished by a core formed

by two P-sheets organized in three-finger loops The first detailed X-ray crystallographic

structure of a cardiotoxin was reported by Rees et al. (47, 48) for cytotoxin Vn 4 from Nuja

mossumbicu mossnnibicn venom. Recently, Bilwes et al. (49) and Sun et al. (50) solved the

structures of toxin y from Nnjn nigricollis venom and cardiotoxin V from Nnju r7nju ufra venom,

respectively. The core of a cardiotoxin is formed by four disulfide bonds with three loops

protruding from this region. There is a large triple-stranded, antiparallel P-sheet connected by

loops I1 and I11 and a short double-stranded P-sheet connected by loop I. Thus, cardiotoxins,

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although differing in primary sequence, have a very similar conformation as viewed by X-ray

crystallography. Although the molecular core containing the three disulfide bonds is rigid, the

three loops appear to be more flexible and variable. In toxin y from Noju tiigricollis venom, loop

I1 appeared to be the most flexible, assuming different conformations in the three different

monomers observed in the crystal asymmetric unit.

The crystal structure of cardiotoxin V is similar to the structure determined by NMR (51)

except for some differences which might be due to different conformational changes in solution

and /or different pH conditions (50). The most significant differences were found in the tips of

loops I and 11, and when the NMR structure of cardiotoxin V is compared to the NMR structure

of toxin y, it appears that the conformational flexibility of loop I1 may be primarily responsible for

the structural differences between the cardiotoxins. There is a wide distribution of charged

residues on the concave side of the dimer which separates the two continuous hydrophobic

columns in the dimer. However, this region could help explain the cell specificity of the

cardiotoxin by allowing for the binding of anionic lipids, carbohydrates or acidic polypeptides

which stabilize the dimer in the membrane.

Whereas toxin y from Nuju nigricollis venom exists as a trimer (49), cardiotoxin V from

Nuju naja afru exists as a dimer (50). In both cases, however, the interaction of the monomers

places the conserved hydrophobic side chains on the “outside” and hydrophilic side chains on the

“inside”. The hydrophobic surface is capable of binding to and inserting into a membrane and the

central hydrophilic surface would permit the passage of ions. Thus, both models are compatible

with the insertion of the multimer into a phospholipid bilayer and the formation of a “pore”

through which small ions may pass.

Function. Only a few studies have examined the effects of cardiotoxins on tissue in idvo, and it is

likely that early studies used cardiotoxin preparations contaminated with a phospholipase.

Cardiotoxins induce prolonged depolarization of skeletal muscle cells in vifro, which leads to

contractions. The rate of depolarization increases with the concentration of cardiotoxin, but even

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MYOTOXINS FROM SNAKE VENOMS 225

at low concentrations (0.15 pM), the cells depolarize (44, 52). Also, different cardiotoxins differ

in their ability to depolarize muscle cells. Hodges et al. (44) found differences in activity of 140-

fold when comparing 18 cardiotoxins.

The ability of cardiotoxins to injure muscle cells was shown by Couteaux et al. (53) and

d’Albis et al. (54) for a toxin from Nuju mossnntbico nmsanibrcu, but these reports did not

describe the mechanism or pathogenesis of myonecrosis. More recently, Ownby et al. (55)

described the myonecrosis induced in viva by cardiotoxin I from Nuju nujn ufru venom. The

toxin was isolated first using fast protein liquid chromatography with a Mono S prepacked HR

10/10 column, then hrther purified with reverse phase HPLC to ensure removal of as much PLA,

contamination as possible. In addition, electrospray mass spectroscopy was used to confirm the

molecular mass of the cardiotoxin.

the first alteration in the skeletal muscle cell was rupture of the plasma membrane in the area of

delta lesions. Myofibnls were hypercontracted and condensed into dense clumps alternating with

Electron microscopic examination of the tissue indicated that

clear areas which contain elements of the sarcotubular system and altered mitochondria. By 24

hours after injection of the toxin, affected cells appeared as empty “bags” containing only

remnants of myofibrils and swollen mitochondria. A cardiotoxin preparation treated with the

PLA, inhibitor, para-bromophenacyl bromide, and demonstrated to have no PLA2 activity on

artificial substrates and cell cultures, induced necrosis of skeletal muscle cells identical to that

caused by the uninhibited cardiotoxin. Additionally, PLA, from the same venom failed to cause

myonecrosis in the same experiment even when used at doses equal to ten times the estimated

contaminating concentration. Thus, it is clear that myonecrosis was due to the cardiotoxin

molecule and not to a contaminant.

Using heavy sarcoplasmic reticulum fractions, Fletcher et al (56) showed that the

contracture of skeletal muscle induced by cardiotoxin from NOJO I ~ O J ~ koozdwu venom was

related to specific induction by the cardiotoxin of Ca” release by the sarcoplasmic reticulum

through a lowering of the threshold for Caz’-induced Ca2’ release On the other hand, the

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mechanism of cardiotoxin-induced hemolysis appeared to be due to a long-term effect on lipid

metabolism. Cardiotoxins also alter Na' channel function (56), activate phospholipase C and

probably tissue PLA, enzymes (57, 58). These actions produce diacylglycerol and free fatty acids.

Since fatty acids also act on Na' channels (59, 60), the release of fatty acids by the sarcolemma-

lysing myotoxins either through their PLA, activity or activation of PLC would lead to an action

on the Na' channels similar to that of myotoxin a and crotamine.

Structure-Function Relationships. The molecular mechanism of action of cardiotoxins remains

unknown, especially the precise way in which these proteins interact with membrane

phospholipids or proteins. However, it is becoming clear from available evidence that these

molecules can penetrate phospholipid bilayers and bring about lysis. Several groups have shown

that the penetration of loop 1 into the hydrophobic core of phospholipid membranes i s responsible

for binding ofthe cardiotoxin to the membrane (46, 61, 62). Loop 1 of the S-type cardiotoxins

and loops 1 and 2 of the P-type cardiotoxins are involved in binding to phospholipids of the

membrane bilayer (46). A general feature of amphiphilic polypeptides is that they go through a

transition between peripheral binding and penetration in the interaction with a membrane. This

allows them to lyse the membrane (63, 64). Sue et al. (65) showed that cardiotoxin A3, a P-type

cardiotoxin, can penetrate and lyse zwitterionic dipalmitoylphosphatidylcholine bilayers forming

small aggregates at a lipidjprotein molar ratio of about 20 in the ripple P,. phase. However, there

is no spectroscopic evidence that a P-sheet cardiotoxin forms a transmembrane component like

the one formed by other amphiphilic a-helix polypeptides such as melittin (65)

Recently, it was found that cardiotoxins bind specifically to heparin and heparin sulfate,

and that this binding potentiates penetration into phospolipid bilayers under physiological ionic

conditions (66). The role of this interaction in myotoxicity has not been investigated.

PHOSPHOLIPASE A, MYOTOXINS

PLA, myotoxins are venom proteins of 12,000 to 16,000 molecular weight which are

structurally homologous to low molecular weight secretory phospholipase A, enzymes Since

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MYOTOXINS FROM SNAKE VENOMS 227

here are several excellent recent reviews of snake venom phospholipase A, enzymes, the current

review will focus only on proteins which have proven myotoxic activity For a more

comprehensive review of the literature on these toxins, the reader is referred to the excellent book

edited by Kini (67) and specific reviews on myotoxins ( 3 , 68, 69).

Structure and Biophysical Aspects. Structurally, the snake venom phospholipase A2 myotoxins

can be classified as either Type I (elapid and hydrophid snake venoms) or Type I1 (viperid and

crotalid snake venoms). Figure 3 shows the amino acid sequences of a few examples of myotoxic

phospholipases from snake venoms separated into Type I and Type I1 categories (for more

sequences see 45) Type I PLA, myotoxins contain 115-120 amino acid residues and seven

disulfide bridges and are similar to pancreatic phospholipase A, enzymes

venom toxins lack the “pancreatic loop” (five extra residues between positions 57 and 58) and the

eight-residue N-terminal pro-protein present in the mammalian pancreatic PLA, enzymes. But,

they contain the “elapid loop” consisting of three residues inserted at positions 52-54 (see Figure

3). Type I1 PLA, myotoxins contain 120-125 amino acid residues and seven disulfide bridges, and

they lack the pancreatic and elapid loops. They also have an additional C-terminal extension which

forms an extra disulfide bridge with a cysteine near the catalytic site. The disulfide bond linking

the P-wing to the N-terminal helix in the Type I enzymes is not present in the Type I1 enzymes

allowing this region of the molecule more flexibility. Some of the Type I1 toxins have a lysine (or

serine) instead of aspartic acid at the 49 position, and Selistre de Araujo et al. (70) proposed a

new family of K49 PLA, toxins as a subgroup of the Type I1 enzymes. The substitution of lysine

for aspartic acid drastically reduced the catalytic activity of the K49 proteins, but there is evidence

from cell culture work that the K49 proteins may actually have lipolytic activity (71 -74). This

important point needs kr ther investigation.

Most of the snake

The crystal structure of several PLA, myotoxins has been solved (75) These proteins all

have a core of three a-helices, a calcium binding site (except for the K49) and a hydrophobic

channel which binds the substrate fatty-acyl chains and mediates the catalysis (76-79). However,

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1 10 20 30 40 50

60 70 80 90 100 110

120

Type I

(a) NLVQFSYLIQCANHGKRPTWHYMDYGCYCGAGGSGTPVDEL[)RCCKIH~DCYDEAGKK-

(a) G C F P K M S A Y D Y Y - C G E N G P Y - C R N I K K K C L , R F V C D C U V E W N I D T K

(a) KRCQ

1 10 20 30 40 50 HLLQFNKMIKFETRKNAIPFYAFYGCYCGWGGRGRPKDATDRCCFVHDCCYGKLAKCN NLFQFEKLIKK-MTGKSGMLWYSAYGCYCGWGGQGRPKDATDRCCFVHDCCYGKVT--- SVLELG~ILQ-ETGKNAITSYGSYGCNCGWGHRGQPKDATDRCCFVHKCCYKKLT---

6 0 70 80 90 100 110 TKYW----DIYRY-SLKSGYITCGKGTWCE~QICECDF~V~ECLR~SLSTYKYGYMFYPD GCNP~DIYTYSV-ENGNIV-CGGTNP-CKKQICECDRAFiAII3FIIDNLLTYDSKTYWKY DCNHKTDRYSYSW-KNKAII-CEEKNP-CLKQMCECUKAVAICLRENLD'TYNKKYKAYF

120 SRCRGPSETC PKNCTKEESEPC KLKCKKPDTC (a) Morech~s sczrrorir Y scii/o/iis. Notcyin. (b) I'ro/nlrrr hrr.w[.s /crn/ictru. Crotosin. (c)Agkr.rrrodr,n piscivorrrs pfscivorirs. D49. (d) AgXislrodon 111 T C ' ~ vorii.s pr.scrvorii.v. K49

Figure 3 . Representative Sequences of Phospholipase A, Myotoxins (Taken from Reference 45)

there is considerable variation in features of surface residues of PLA, nlyotoxins from different

sources ( 7 5 )

These proteins have been detected in venom as monomeric, dimeric and multimeric forms.

Notexin, pseudexin and nigroxin, Type 1 PLA, myotoxins are all present as monomers in active

form. However, taipoxin and textilotoxin, boih Type I enzymes, consist of multiple proteins non-

covalently bound The only Type I enzyme known to exist as a dimer with covalently bound

subunits is P-bungarotoxin, and it has been shown to lack inyotoxic activity (70. 74), although it

has strong neurotoxic activity Of the D49 Type 11 enzymes that are known to be myotoxic,

crotoxin and niojave toxin are known to be composed of two different polypeptides non-

covalently bound, whereas Bothrops asper inyotoxin Ill (80), lipern r.u.c-s.e//i VRV-PL-VIlIa

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MYOTOXINS FROM SNAKE VENOMS 229

(81) and Crofalzis viridis viridis C W myotoxin (82) are all single chain polypeptides. Of the

K49 Type II enzymes known to be myotoxic by histological assay, some appear to be monomeric

whereas others are dimeric. Agkistrodonp. piscivonrs K49 PLA, (83 , 84) and Agkistrodon

conforfrix laticincfus K49 PLA, (85) are both monomers as determined by their crystal structure.

In contrast, B. arper myotoxin I1 is a dimer in its crystal state (86). It is not clear yet what r& if

any, the subunit structure might play in the myotoxic activity of these proteins However, it is

probably not a strict requirement since some of the myotoxins consist of only one polypeptide

chain and have sufficient activity to induce myonecrosis. This is true for bothType I and Type II

PLA, myotoxins It is possible that these proteins could exist as monomers in solution yet form

multimers upon interaction with the surface of a membrane.

Functioh. Although the structures of phospholipase A, myotoxins differ, the pathogenesis of

myonecrosis they induce appears to be the same, at least as far as can be measured with current

assays. This topic has been extensively reviewed (1-4,68, 74) and thus will not be repeated here.

Briefly, upon experimental injection these toxins act very rapidly to cause necrosis within 5 min of

an intramuscular injection The predominant pattern of necrosis is characterized by the presence

of “delta lesions” and highly contracted and clumped myofilaments which alternate with clear

areas of cytoplasm. Delta lesions are wedge-shaped clear areas in the muscle cell defined by a

ruptured sarcolemma at their base and an area of cytoplasm devoid of organelles extending into

the interior of the cell. By 24-48 hours after injection, most of the affected cells have a less-

densely clumped appearance and contain phagocytic cells. By 72 hours small myogenic cells are

present in the sarcolemmal tubes, and by 3-4 weeks mature muscle cells can be observed,

although the nuclei remain centrally located. This pathogenesis is remarkably similar to that

induced by the cardiotoxin from Nuju nuju nuju venom (59, indicating that structurally different

toxins can induce a similar pathogenesis of myonecrosis. The general consensus is that the

sarcolemma is the initial site of action of these toxins, but the exact mechanism of action is not

known.

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Structure-Function Relationships. Identification of specific amino acid residues involved in the

action of PLA, myotoxins is still incomplete. Kini and Iwanaga (40) proposed that the pre-

synaptically neurotoxic PLA, toxins with myotoxic activity all contained a characteristic charge

density distribution pattern with a cationic site around residues 79-87 having the pattern

+OO+++OO+. However, they also point out that crotoxin, a potent myotoxin, does not have this

region. Also, many of the non-neurotoxic PLA, myotoxins such as Bo/hrops mper myotoxin I1

and 111 and Bo/hropsjnrnrocimrr bothropstoxin I do not contain this site (68). This site may play

a role in the activity of some myotoxins, but it must not be essential since many myotoxins do not

have it.

The N-terminal region appears to play some role in myotoxicity since removal of an N-

terminal octapeptide from B. usper myotoxin I1 reduces the myotoxic activity to one-third of the

original (87). This is understandable since there is considerable evidence that this region is

important in interfacial recognition and adsorption. Recent studies on the D49 PLA- , f rom

Agkistrodoripiscivorrispisch,orra venom (88) indicate that Lys-7 and Lys-10 are critical for

productive adsorption to anionic interfaces Interestingly, the topographic location of these

residues in other enzymes may be quite different indicating that the arrangement of these lysines is

species-specific. Selistre de Araujo et al. (70) proposed that some conserved residues, i.e , K7,

E12, T13 and K15, at the N-terminus ofthe K49 PLA, myotoxins play a role in myotoxicity.

These residues are also present in some D49 PLA, myotoxins. However, in the K49 PLA,

myotoxin, B. usper myotoxin 11, this region forms part of a homodimer interface (86).

presumably making it inaccessible to the membrane.

The role of the calcium-binding loop (residues 25-33) in myotoxicity is not clear,

especially for the K49 PLA, myotoxins in which it is not conserved. However, it may be

important because of its role in catalytic activity of the D49 PLA, myotoxins. Likewise, the role

of the Class I loop (residues 55-67) in myotoxicity is not clear. This loop is clearly important in

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MYOTOXINS FROM SNAKE VENOMS 23 1

catalytic activity of these enzymes, and thus may play a role in myotoxicity related to phospholipid

hydrolysis.

Selistre de Araujo et al. (70) proposed a role for some conserved residues in the 0-wing

(residues 74-84), i.e., K78 and K80. In the K49 PLA, myotoxins, these residues are spatially

closely related to the proposed myotoxic site in the N-terminal region, and could thus be

important for binding to the target site. However, in B. usper myotoxin 11, a K49 PLA,,

residues in this region form part of a homodimer interface (86).

There is considerable evidence for involvement of the C-terminus (residues 11 5-134) in

cytotoxicity, if not myotoxicity. This region is rich in lysines and is known to bind heparin and

other considerable experimental evidence confirms its role in cytotoxicity (89). K115 and K116

in this region are part of the myotoxic site proposed by Selistre de Araujo et al. (70). However, in

crotoxin and mojave toxin, both potent myotoxins, this region is completely different from that of

the K49 PLA, myotoxins (70). Thus, the C-terminus alone cannot be responsible for myotoxic

activity.

It is possible that the N-terminal is responsible for interfacial recognition and binding; the

C-terminal on some molecules binds to heparin at the surface to further assist the toxin in

penetrating the bilayer and binding the toxin to the membrane. Penetration occurs by virtue of

the cationichydrophobic site and the membrane is disrupted. This mechanism could explain the

interaction between the toxin and the phospholipid membrane which could lead to lysis, but it

does not appear to explain the specificity of these toxins. Numerous studies have shown that the

PLA, myotoxins have some degree of specificity for the muscle cell membrane. This has led to

the postulation of the involvement of specific target molecules in the membrane, either

phospholipid or protein. Two protein receptors have been identified for a single-chain neurotoxic

PLA, (OS,), one in brain membranes (N-type) and one in muscle cells (M-type) (90). It is not

known whether either of these serve as a receptor for any myotoxic PLA,.

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STRUCTURE-FUNCTION RELATIONSHIPS OF MYOTOXINS

Snake venom myotoxins induce either a rapid necrosis of skeletal muscle cells,

characterized by lysis of the sarcolemma (cardiotoxins and phospholipase A, tnyotoxins), or a

slower necrosis characterized by dilatation of sarcoplasmic reticulum and lack of sarcolemmal

lysis (myotoxin a, crotamine) In both cases, however, the plasma membrane or sarcolemma of

the muscle cell appears to be the initial site of action, but it is still not clear whether any of these

toxins are internalized The myotoxins may bind to a protein receptor as yet unidentified There

is some evidence that the small, basic polypeptide myotoxins and cardiotoxins bind to the Na'

channel The PLA, myotoxins may bind to the M-type PLA, receptor There is also evidence for

electrostatic interaction between positively charged groups on the myotoxins and negatively

charged phospholipids All of the known myotoxins have a highly positively charged surface, and

the presence of cationic residues closely positioned to hydrophobic residues in the toxin molecule

seems to play a role in their myotoxicity Perhaps ongoing site-directed mutagenesis studies will

reveal the exact residues involved in the myotoxic action. Also, all of these molecules have

considerable P-sheet structure, and very little has been done to define the interaction of P-sheet

structure with phospholipids or proteins in membranes Some are known to bind to specifically to

heparin and heparin sulfate, and the role of this binding in myotoxicity needs to be investigated

Finally, although they all have some molecular features in common, and they all appear to act

initially on the sarcolemma, they may actually have different molecular sites and their mechanism

of action on the muscle cell may be different However, at least in the case of the cardiotoxins

and the phospholipase A2 myotoxins, the pathologic response ofthe muscle cell appears to be

essentially identical. Future studies employing the techniques of site-directed mutagenesis, NMR,

and more precise procedures for defining the interaction between these toxins and the muscle cell

should better define the molecular myotoxic site of the snake venom myotoxins as well as the

exact mechanism by which they damage and kill skeletal muscle cells.

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MYOTOXINS FROM SNAKE VENOMS 233

ACKNOWLEDGEMENTS

The author thanks the College of Veterinary Medicine, Oklahoma State University for continuous

support; Terry R. Colberg and Jason Evans for critically reading the manuscript; and Drs. Heloisa

S. Selistre de Araujo and Jeffrey E Fletcher for fruithl collaboration and stimulating discussions.

1 .

5.

6 .

7.

8.

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from Crolalics viridrs coticolor venom. Toxicoti 25, 677-680. 12. Aird, S.D., Kruggel, W.G. and Kaiser, 1.1. (1991) Multiple myotoxin sequences from the

venom of a single prairie rattlesnake (Cro/nlris iwrdis iiridis). Toxicoti 29, 265-268. 13. Nedelkov, D. And Bieber, A.L. (1997) Detection of isoforms and isomers of rattlesnake

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