study of the mechanism of action of snake...
TRANSCRIPT
Sede Amministrativa: Università degli Studi di Padova
Dipartimento di Biologia
SCUOLA DI DOTTORATO DI RICERCA IN BIOSCIENZE E BIOTECHNOLOGIE
INDIRIZZO BIOLOGIA CELLULARE
CICLO XXVI
STUDY OF THE MECHANISM OF ACTION OF SNAKE MYOTOXINS
Direttore della Scuola : Ch.mo Prof. Giuseppe Zanotti
Coordinatore d’indirizzo: Ch.mo Prof. Paolo Bernardi
Supervisore: Ch.mo Prof. Cesare Montecucco
Dottorando: Julián Fernández
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Acknowledgements
I would like to thank Prof. Cesare Montecucco for his guidance, help and for the
opportunity to work in his lab. I am also grateful to Dr. Fiorella Tonello for her advice and
suggestions, and to my labmates and academic staff (Sadia, Domenico, Maria, Elisa,
Marco, Irene, Oneda, Samuele, Veronica, Lydia, Paola, Michele, Morena, Michela, Ornella)
for helping me when I was in need.
To Bruno, Chema and Yami, for all their help.
I am grateful also to Anthony Postle and Grielof Koster from the Infection,
Inflammation and Immunity division, at the School of Medicine of the University of
Southampton.
I would like to thank my family for their love and support.
Finally, thanks to the CARIPARO Foundation for the scholarship and to the
University of Padova and University of Costa Rica for their support.
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Index
Sommario........................................................................................................10
Summary.........................................................................................................12
1. Introduction................................................................................................14
1.1 Snake bite: a global neglected condition.......................................14
1.2 The burden of snake bite...............................................................15
1.3 Clinical features and treatment of envenomings by B. asper........17
1.4 Bothrops asper snake venom composition....................................19
1.5 Snake venom phospholipases A2 (PLA2s).......................................20
1.6 Myotoxins.....................................................................................23
1.7 Cellular and molecular pathology induced by myotoxins………..….26
2. Objectives....................................................................................................30
3. Chapter 1: Muscle phospholipid hydrolysis by Bothrops asper Asp49 and
Lys49 phospholipase A₂ myotoxins--distinct mechanisms of action….....….31
4. Chapter 2: Why myotoxin-containing snake venoms possess
powerful nucleotidases?…………………………………………………….………………...….43
5. Chapter 3: Ongoing experiments…...............................................................49
6.Concluding remarks…....................................................................................63
7. References....................................................................................................65
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Table list Page
Table 1. Composition of venoms from adult specimens of B. asper 19
from the Caribbean and the Pacific versants of Costa Rica, according
to protein families.
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Figure list Page
Figure 1. Distribution of the Bothrops asper–atrox complex 16
in Central and South America.
Figure 2. Adult B. asper specimen. 18
Figure 3. Comparison of group I and group II snake venom PLA2s. 21
Figure 4. Structural classification of snake venom myotoxins. 24
Figure 5. Summary of the proposed models for the "toxic site" 26
of Lys 49 myotoxins.
Figure 6. Effects of myotoxic snake PLA2 s and PLA2 homologues 27
in skeletal muscle cells.
Figure 7. Purification of fluorescent Mt-I by RP-HPLC 57
Figure 8. Localization of the conjugated ZQG-fluorophore in MT-I. 58
Figure 9. Comparison of the toxic activity of Mt-I and the 59
fluorescent Mt-I in contact with C2C12 myotubes.
Figure 10. Visualization of fluorescent Mt-I (25 µg/ml) 60
in contact with primary mouse myotubes.
Figure 11. Binding of fluorescent Mt-I to the membrane 61
of tibialis anterior muscles.
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Figure 12. Damaged tibialis anterior muscle fibers after 62
30 min of incubation with the fluorescent Mt-I.
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Abbreviations
Asp Aspartic acid
ATP Adenosine triphosphate
Ca2+ Calcium
DMEM Dulbecco's modified Eagle medium
FA Fatty acid
Lys Lysine
lysoPA Lysophosphatidic acid
lysoPC Lysophosphatidylcholine
lysoPE Lysophosphatidylethanolamine
lysoPS Lysophosphatidylserine
Mt-I Myotoxin-I from Bothrops asper venom
Mt-II Myotoxin-II from Bothrops asper venom
PA Phosphatidic acid
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PLA2 Phospholipase A2
PS Phosphatidylserine
RP-HPLC Reverse phase high performance liquid chromatography
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Sommario
Gli avvelenamenti da morso di serpente costituiscono un problema molto
importante di sanità pubblica nelle regioni rurali di Africa, America Latina e Asia. I serpenti
del genere Bothrops sp. sono la causa della maggior parte dei morsi di serpente in America
Latina. Le fosfolipasi A2 (PLA2s) sono parte dei principali componenti dei veleni di questo
genere, e la miotossicità è uno dei loro effetti tossici più importanti. Il meccanismo
d’azione con cui queste proteine danneggiano le cellule muscolari non è ancora stato del
tutto chiarito.
Nella prima parte di questo lavoro, è stata fatta un’analisi in spettrometria di
massa dei prodotti da idrolisi causati dall’intossicazione con miotossina I (Mt-I) e
miotossina II (Mt-II) di Bothrops asper utilizzando miotubi C2C12 in cultura, e
dall’iniezione ex vivo di Mt-I, Mt-II e veleno di B. asper nei muscoli tibialis anterior di topi
CD-1. La Mt-I e il veleno di B. asper hanno indotto un aumento consistente di
lisofosfatidilcolina (lysoPC) e lisofosfatidiletanolamina (lysoPE) nei campioni analizzati.
Invece, la Mt-II non ha alterato significativamente la concentrazione di nessun fosfolipide.
Questi risultati mostrano per la prima volta che la Mt-I e il veleno di B. asper causano una
produzione significativa di lisofosfatidilcolina e lisofosfatidiletanolamina quando vengono
iniettati nel muscolo. I prodotti principali della loro azione sonno lysoPC e lysoPE, e la
proporzione di idrolisi di fosfatidilserina e questi due lipidi indica che il sito principale
d’azione de la Mt-I è la parte esterna della membrana plasmatica. La Mt-II anche se
induce una entrata di calcio dentro alle cellule muscolari non provoca un’attivazione
significativa delle fosfolipasi endogene.
Nella seconda parte, è stata quantificata l'attività ATPasica, ADPasica e
nucleotidasica dal veleno di B. asper, ed è stato analizzato il ruolo di queste attività nei
veleni di serpenti contenenti miotossine. Partendo dal fatto che le miotossine inducono il
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rilascio di ATP in vivo, la presenza di nucleotidasi nel veleno porta ad una elevata
concentrazione di adenosina in circolo, che ha attività ipotensiva, paralizzante e
anticoagulante.
Nella terza parte è stata messa a punto una reazione enzimatica di coniugazione
della Mt-I con un derivato peptidico tramite l'enzima transglutaminasi. La reazione di
coniugazione ha permesso di ottenere la tossina con una modifica ad un singolo amino
acido e, dai primi esperimenti di caratterizzazione, con attività tossica e fosfolipasica
ancora presenti. Lo scopo finale è di coniugare la Mt-I con un fluoroforo, per studiare la
localizzazione della tossina quando viene aggiunta ai miotubi in cultura o iniettata nei
muscoli tibialis anterior di topi. Successivamente di coniugare la tossina con un derivato
della biotina per isolare dalle cellule le proteine od altre molecole interagenti con essa.
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Summary
Envenoming due to snake bite is a neglected disease that is a very important public
health problem in rural regions of Africa, Latin America and Asia. Snakes from the genus
Bothrops cause most of the snake bites in Latin America. Phospholipases A2 (PLA2s) are
major components of the venoms from this genus, exerting myotoxicity as one of their
most relevant toxic actions. Many parts of the mechanism of action by which these
proteins damage muscle cells are not known. Therefore, it is crucial to further study these
toxins.
In the first part of this work, the measurement of the hydrolysis of phospholipids
caused by the addition of myotoxin I (Mt-I) and myotoxin II (Mt-II) from the venom of
Bothrops asper to C2C12 myotubes, and the ex vivo injection of the venom of B. asper,
Mt-I, and Mt-II in tibialis anterior muscles of CD-1 mice was performed. Results show that
Mt-I and B. asper venom caused an increase in lysophosphatidilcholine (lysoPC) and
lysophosphatidilethanolamine (lysoPE) species, while Mt-II generated no significant
production of lysophospholipids of any of these species. These results show for the first
time that Mt-I and B. asper venom are able to hydrolyze phospholipids when injected in
muscles. Their main products of hydrolysis are lysoPC and lysoPE, and their proportions
indicate that the main site of action of Mt-I is the external part of the plasma membrane.
Mt-II, which causes Ca2+ entry into muscle cells, does not cause a significant activation of
the endogenous PLA2s in injected muscles.
ATPase, ADPase and nucleotidase activities of B. asper venom were quantified in
the second part, and the role of these activities in venoms containing myotoxins was
analyzed. Since myotoxins release ATP in cultured myotubes as well as in vivo, there is a
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combined action with ATPase, ADPase and nucleotidase activities in the venom that
creates a high concentration of adenosine, which has hypotensive, paralyzing and anti-
coagulant activities.
In the third part, which contains the ongoing experiments, a conjugation reaction
of Mt-I with peptide derivatives using a transglutaminase enzyme has been developed.
Mt-I was modified in a single amino acid, with a good percentage of catalytic and toxic
activity still present. The aim is to obtain a Mt-I conjugated to a fluorophore to study the
localization of the toxin in cultured myotubes or when injected in mouse tibialis anterior
muscles. Subsequently, Mt-I will be conjugated to a biotin derivative as a tag to isolate
the toxin after cell intoxication together with interacting proteins or other molecules.
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1. Introduction
1.1. Snake bite: a global neglected condition
Envenomings resulting from snake bite represent one of the serious public health
problems in low and middle income countries, mainly in rural regions of Africa, Latin
America and Asia (Gutiérrez et al., 2006). Since most of the poorest communities in the
world live in these regions, there is a high morbidity and mortality due to the lack of
access to health services and scarcity of antivenom (Theakston and Warrell, 2000).
The relation between poverty, snake bite mortality and government expenditure in
health has already been studied and therefore snake bite has been defined as a disease of
the poor (Harrison et al., 2009). Sadly, the huge affliction caused by this problem remains
unrecognized to the global public health authorities. This is clearly shown by the fact that
it entered WHO’s list of neglected tropical diseases as late as April, 2009 (Williams et al.,
2010). Later, in 2011, it was changed to a neglected condition. One of the main reasons
for this lack of recognition is the insufficient global epidemiological data (Warrell, 2010),
which makes it very difficult for public health authorities to grasp the actual impact of this
disease and, consequently, to improve the prevention and treatment of snake bites
(Kasturiratne et al., 2008).
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1.2 The burden of snake bite
There are at least 600 species of venomous snakes in the world
(http://apps.who.int/bloodproducts/snakeantivenoms/database/). These venomous
snakes are distributed almost everywhere in the world, including many oceans, with the
exception of some islands, frozen environments and high altitude zones (Kasturiratne et
al., 2008). The most relevant toxins in human envenoming are those that alter the
nervous, cardiovascular, and haemostatic systems, and those that cause tissue necrosis
(Warrell, 2010).
Not enough accurate data about snake bites are available because the areas where
snake bites are more common are the rural parts of tropical developing countries. For this
reason, snake bite is likely to be under-reported (Warrell, 2010). The first attempt to
describe the global magnitude of this problem was elaborated by Swaroop and Grab in
1954, who estimated a global total of 30 000 – 40 000 deaths from snake bite per year.
Then, Chippaux in 1998 estimated a total of 5 400 000 bites, 2 500 000 envenomings, and
125 000 deaths per year in the world. In 2008, Kasturiratne et al. estimated that every
year, between 1.2 and 5.5 million snake bites could occur and at least 421 000
envenomings and 20 000 deaths from snake bite could occur.
The highest burden of snake bite in the world was estimated in South and
Southeast Asia, sub-Saharan Africa, and Central and South America (Chippaux, 1998;
Kasturiratne et al., 2008). India had at least 11 000 deaths per year, making it the country
with the highest number of fatalities due to snake bite in the world. Bangladesh and
Pakistan had over 1,000 deaths per year (Kasturiratne, et al., 2008). In the case of Central
and South America, despite having about 25% of the global snake bite incidence, the
mortality due to snake bite was lower than in other high incidence regions, possibly
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because of better snakebite management systems in many Latin American countries
(Kasturiratne, et al., 2008).
A large proportion of the victims that survive a snake bite are affected by
permanent physical and psychological consequences. The main cause of permanent
disability in snake bite survivors is local necrosis (Warrell, 2010). In the case of large areas
of skin necrosis, debridement and grafting are necessary. If there is destruction of deep
tissues, amputation might be needed (Warrell, 2010).
The majority of severe cases of snake bite envenoming are provoked by species of
the families Elapidae and Viperidae. The highest number of bites and fatalities are caused
by the species Echis sp. (saw-scaled vipers) in Northern Africa, B. asper and B. atrox (lance-
headed pit vipers) in Central and South America (Figure 1), and Naja sp. (cobras) and
Bungarus sp. (kraits) in Asia (Gutiérrez et al., 2006).
Figure 1. Distribution of the Bothrops asper–atrox complex in Central and South America.
B. asper is shown in closed circles, B. atrox in gray, and B. colombiensis in white and
yellow. Taken from Alape-Girón et al. (2009).
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In Latin America, the incidence of snake bite envenomings ranges from 5 to 62
cases / 100 000 population per year, depending on the country (Chippaux, 2006; Otero,
2009). Panama is the country with the highest incidence of snake bites in the region, most
of them caused by B. asper. Also in Costa Rica, Nicaragua, Honduras, Panama, Colombia,
Venezuela and Ecuador there is a high proportion of snake bites caused by B. asper. In
fact, it has been estimated that 50–80% of the snake bites and 60–90% of deaths
secondary to snake bites in Central America and northern South America are caused by
this species (Chippaux, 2006; Otero, 2009).
1.3 Clinical features and treatment of envenomings by B. asper
B. asper, a widely distributed pitviper in Central America and northern areas of
South America (Figure 1), is an irritable species that is able to inflict bites in any
anatomical region (Sasa et al., 2009). Clinical features after envenomations caused by this
species are similar in Central and South America. Edema is present in 95% of the cases,
and is detectable as early as 5 min after the bite. Local hemorrhage (34% of cases), is
present during the first 5–30 min. Blisters (12% of cases), dermonecrosis and myonecrosis
(10% of cases) are also important manifestations. Defibrinogenation is the classic sign of
systemic envenoming and occurs in 60–70% of cases, usually within 30–60 min.
Alterations such as thrombocytopenia (15–30% of cases), hypotension (10–14%) and
systemic bleeding (25–30%) are also observed (Otero et al., 1992, 2009).
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Figure 2. Adult B. asper specimen (A) Head of a female
(B) Defensive posture, adopted when the snake is cornered.
Figure taken from Sasa et al. (2009).
The severity of local envenoming is defined by the level of edema and the presence
of necrosis, while the severity of systemic envenoming is defined by the levels of
alteration of blood coagulation, bleeding and presence of life-threatening complications
such as shock, acute renal failure and damage of vital organs (Otero, 2009). It has been
estimated that 15% of the victims of a snake bite by B. asper present severe systemic
envenoming and 10% of the victims suffer severe local envenoming (necrosis and edema
extending to the trunk). Patients with severe envenoming are at risk of death and/or
permanent sequelae (Otero et al., 1992).
The scientifically validated treatment of snake bites is the intravenous
administration of animal-derived antivenoms. Horse and sheep are the animals used most
frequently to produce antivenoms, after immunization with the appropriate venoms
(Gutiérrez et al., 2006). There are laboratories, in all the continents, that are in charge of
the production of antivenoms, which consist of whole IgG molecules or products of their
enzymatic digestion such as bivalent F(ab')2 or monovalent Fab. Since there is a great
immunochemical diversity in the toxins present in snake venoms from different species,
the efficacy of antivenoms is generally restricted to a limited geographical and biological
spectrum (Gutiérrez et al., 2006).
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1.4 B. asper snake venom composition
The venom of B. asper contains proteins grouped in 8 families (Table 1):
metalloproteinase, serine proteinase, C-type lectin-like, L-amino acid oxidase, disintegrin,
DC-fragment, cysteine-rich secretory protein, and phospholipase A2 (Alape-Girón et al.,
2008). At least 25 proteins from this venom have been isolated and characterized (Angulo
and Lomonte, 2009).
Table 1. Composition of venoms from adult specimens of B. asper from the Caribbean
and the Pacific versants of Costa Rica, according to protein families. Taken from Alape-
Girón et al. (2009).
It has been estimated that phospholipases A2 are a major component of the venom
of B. asper (Table 1). These proteins comprise 28.8% (B. asper from the Caribbean region
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of Costa Rica) to 45.1% (B. asper from the Pacific region of Costa Rica) of total venom
proteins (Alape-Girón et al., 2008).
1.5 Snake venom phospholipases A2 (PLA2s)
Phospholipases A2 (PLA2s) are widely distributed in nature. They are found in large
amounts in the pancreas of mammals and in the venom of snakes, bees, scorpions
and lizards (Schaloske and Dennis, 2006). PLA2s are the major component in the venom
of many snakes, such as B. asper. In a study by Alape-Girón et al. (2008), 45% of the
proteins from B. asper venom (specimens from the Pacific versant of Costa Rica) were
PLA2s.
PLA2s are enzymes that catalyze the hydrolysis of the ester linkage of the C2 of 3-
sn-phosphoglycerides, producing lysophospholipids and free fatty acids in a calcium-
dependent reaction. Enzymatically-active PLA2s require calcium for the stabilization of
their catalytic conformation. The binding site for calcium is formed by carbonyl groups of
Tyr 28, Gly 30 and Gly 32 and the carboxyl group of Asp 49. Two water molecules
structurally complete the calcium-binding sphere, so that a pentagonal bipyramid is
formed (Scott et al., 1990). The characteristic structure of PLA2s from Bothrops sp. consists
of three alpha helices, a beta sheet, a short helix and a calcium binding loop (Arni and
Ward, 1996). Snake venom PLA2s have neurotoxic, myotoxic, hemorrhagic, hemolytic,
necrotic, and edema-inducing activities (Kini, 2003; Gutiérrez and Lomonte, 1997). Some
of these PLA2s can induce or inhibit platelet aggregation, or both, through a biphasic
response (Kini and Evans, 1997).
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The superfamily of PLA2 enzymes currently consists of 15 groups. There are 5
different types of enzymes in these groups: the secreted PLA2s, the cytosolic PLA2s, the
calcium-independent PLA2s, the acetylhydrolase-platelet activating factor and the
lysosomal PLA2s. Assignment to a particular group is based on the mechanism of catalysis,
and structural and functional characteristics. Snake PLA2s are classified within the group of
small (12-15 kDa), secreted PLA2s. PLA2s of elapids are located in the group IA, whereas
those of viperids are located in the group II (Schaloske and Dennis, 2006). The comparison
of PLA2s of elapids and viperids reveals a conserved three-dimensional structure.
However, as shown in figure 3, they differ in the position of one of their seven disulfide
bonds, and in their C-terminal regions (Lomonte and Gutiérrez, 2011).
PLA2s of B. asper, which belong to the family Viperidae, are located in subgroup IIA
(Schaloske and Dennis, 2006).
Figure 3: Comparison of group I and group II snake venom PLA2s. In (a), notexin
(PDB: 1AE7), a group I PLA2 from the elapid snake Notechis s. scutatus is shown. In (c), D49
PLA2 (PDB: 1VAP), a group II enzyme from the viperid snake Agkistrodon p. piscivorus is
shown. There is an overall structure conservation, as shown in (b), with the C-terminal
sequence of the group II enzymes inside an oval, and the“elapid loop”of group I
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enzymes evidenced with an arrow. Taken from Lomonte and Gutiérrez (2011) and
Montecucco et al. (2008)
In the IIA subgroup, PLA2s are further divided into 2 groups (Lomonte et al., 2003):
1) Asp49 PLA2s, which have an aspartate residue at position 49 and have catalytic
activity.
2) PLA2 homologues: The most common are Lys49, but there are Ser49, Arg49,
Gln49 and Asn49. These proteins have, respectively, a lysine, serine, arginine,
glutamine, or asparagine residue at position 49 and have no catalytic activity
(Lomonte et al., 2003). Due to their high structural similarity with classical Asp49
PLA2s, these proteins have been referred to as “PLA2 homologues” (Lomonte and
Gutiérrez, 2011).
PLA2s can also be classified according to their isoelectric point as acidic, basic or
neutral phospholipases. There is a tendency towards a higher toxicity of basic PLA2s, in
comparison with their acidic counterparts (Lomonte and Gutiérrez, 2011). The presence of
different isoforms of PLA2s in snake venoms has been demonstrated, either in the venom
of one species or in the venoms of snakes of the same genus (Santos-Filho et al., 2008 and
Alape-Girón et al., 2008). In the venom of B. asper, basic PLA2s are more abundant than
acidic PLA2s (Alape-Girón et al., 2008; Fernández et al., 2010).
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1.6 Myotoxins
Many basic Asp49 PLA2s and Lys49 “PLA2-like” proteins have shown myotoxic
properties, regardless of their ability to catalyze the hydrolysis of phospholipids. Lys49
“PLA2-like” proteins can therefore be used as models for the induction of myonecrosis by
a mechanism of action that is independent of catalytic activity (Lomonte et al., 2003).
Myotoxins can be defined as natural components of venom secretions that induce
irreversible damage to skeletal muscle fibers (myonecrosis) upon injection into higher
animals (Lomonte et al., 2003). Myotoxins can additionally cause neurotoxicity, edema,
hypotensive and proinflammatory effects and in vitro anticoagulant and platelet-
modulating effects (Gutiérrez et al., 1986; Angulo and Lomonte, 2009), together with the
induction of cytokines (Lomonte et al., 1993), and cytotoxic activity (Lomonte et al., 1994).
These toxins may act locally or systemically and they can be structurally classified
in three groups: small myotoxins, three-finger toxin cytolysins and PLA2s (Figure 4):
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Figure 4: Structural classification of snake venom myotoxins. These toxins can be
classified in three groups: small myotoxins, three-finger toxin cytolysins and
phospholipases A2. Figure taken from Lomonte and Rangel, 2012.
The venom of B. asper contains both Asp49 PLA2s and Lys49 “PLA2-like” proteins.
B. asper myotoxins I, III, and the PLA2 reported by Mebs and Samejima (1986) are Asp49
isoforms, while myotoxins II and IV are Lys49 PLA2 homologues. The basic PLA2s of B.
asper have 121–122 amino acid residues, with 14 Cys residues and seven disulfide bridges
(Kaiser et al., 1990; Francis et al., 1991). They are not glycosylated, and form stable,
noncovalently associated homodimers in their native state (Lomonte and Gutiérrez, 1989;
Francis et al., 1991). Only the three-dimensional structure of Myotoxin II (Mt-II) has been
determined, by Arni et al. (1995) and Murakami et al. (2005) at resolutions of 2.8 Å and
1.7 Å, respectively. The resolved structure of BthTx-II from the venom of Bothrops
jararacussu (Correa et al., 2008), a PLA2 which has a sequence identity of 91% to myotoxin
I (Mt-I) from B. asper, is used to obtain the three-dimensional model of Mt-I.
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The toxic effects of Asp49 myotoxins have been attributed mainly to their
phospholipase A2 activity, either because phospholipid hydrolysis promotes membrane
instability or because the lysophospholipid and fatty acid products have toxic effects
(Lomonte and Gutiérrez, 2011). However, toxicity and catalytic activity are poorly
correlated, since some highly toxic PLA2s have a very low catalytic activity, while some
highly active enzymes are non-toxic or have a low toxicity. Their catalytic activity is very
important for their toxicity, and if this activity is inhibited, myotoxicity is also greatly
reduced or eliminated (Lomonte and Gutiérrez, 2011). The interfacial binding properties
of these PLA2s toward their substrates also constitute a very important component in their
mechanism of action (Kini and Evans, 1989). The possibility of the existence of a
catalytically-independent mechanism of toxicity in these toxins, additional to their
catalytic-mediated actions, still needs to be further studied and clarified.
PLA2-like Lys49 toxins are not able to hydrolyze phospholipids but still cause toxic
effects. The main toxic site of these toxins, according to most approaches, consists of a
short sequence rich in cationic and hydrophobic residues that is located near the C-
terminus of these proteins (Figure 5). This cationic C-terminal region interacts with anionic
components of cell membranes and causes the loss of permeability control and an influx
of calcium that ultimately leads to necrotic cell death (Montecucco et al., 2008; Gutiérrez
and Ownby, 2003). The precise biophysical mechanism by which Lys49 myotoxins disrupt
the sarcolemma is still unknown, but it could involve a partial insertion of amino acids
from the toxic site of myotoxins into the phospholipid bilayer (Rangel et al., 2011), or a
disorganization of the sarcolemma through phospholipid extraction (de Oliveira et al.,
2009a), or both (Lomonte and Rangel, 2012). The identity of the "acceptor" of Lys49 toxins
in the sarcolemma is still not known, although Mt-II has shown significant binding to
phospatidic acid (PA) and phosphatidylserine (PS), two anionic phospholipids, under in
vitro conditions (Lomonte and Rangel, 2012). Lys49 myotoxins have also shown high
affinity binding to the VEGF-R (Yamazaki et al., 2005), but a study has shown no functional
26
relevance of VEGF-R in the mechanism of action of Lys49 myotoxins (Lomonte and Rangel,
2012).
Figure 5. Summary of the proposed models for the "toxic site" of Lys 49 myotoxins. Model
(A) was proposed by Lomonte et al., 1994a, model (B) by Selistre de Araujo et al., 1996
model (C) by Chioato et al., 2002 and model (D) by dos Santos et al., 2009. Modified from
Lomonte and Rangel, 2012
1.7 Cellular and molecular pathology induced by myotoxins
The action of myotoxins on skeletal muscle is summarized in Figure 6. The toxins
are able to bind unknown receptors or acceptors in the outer part of the sarcolemma,
where Asp49 myotoxins immediately begin to hydrolyze phospholipids and generate
lysophospholipids and fatty acids, disrupting the cell membrane. Lys49 myotoxins disrupt
the cell membrane through a mechanism that needs further characterization (Montecucco
et al., 2008; Lomonte and Rangel, 2012). As a consequence, both toxins cause a massive
calcium influx (Cintra-Francischinelli et al., 2009) that leads to hypercontraction of
27
myofilaments which further damages the sarcolemma. Calcium influx also causes the
activation of calpains (which degrade cytoskeletal components), mitochondrial damage,
and ultimately necrotic cell death (Montecucco et al., 2008; Gutiérrez and Ownby, 2003).
The loss of membrane permeability causes the release of ATP from damaged cells, which
acts as a "danger signal" and causes further damage by its action on purinergic receptors
(Cintra-Francischinelli et al., 2010). K+ is also released and, together with the release of
ATP, causes the pain sensation in the victim (Cintra-Francischinelli et al., 2010).
Figure 6. Effects of myotoxic snake PLA2s and PLA2 homologues in skeletal muscle cells.
Myotoxins bind to either high-affinity specific protein receptors (R) or to low-affinity lipid
28
domains of the plasma membrane (Step 1). Hydrolysis of phospholipids is caused by
catalytically active myotoxins (Step 2), which alters the plasma membrane. As shown in
step 3, enzymatically-inactive myotoxins act via direct perturbation of the membrane.
There is a large increase in the concentration of cytosolic Ca2+ (Step 4) caused by both
types of myotoxins, which induces hypercontraction of myofilaments (step 5) and
therefore may cause mechanical damage to the plasma membrane. Ca2+ uptake by
mitochondria causes mitochondrial swelling, disorganization of cristae, formation of
hydroxyapatite crystals, flocculent densities, and also it was speculated to cause the
opening of the permeability transition pore (step 6). Ca2+-dependent proteinases
(calpains) could also be activated, causing the degradation of cytoskeletal components
(step 8). Ca2+-dependent cytosolic PLA2s could promote further hydrolysis of intracellular
membranes and plasma membrane (step 9). Damage to the plasma membrane could also
allow the entry of myotoxic PLA2s (step 10), which could hydrolyze and damage
intracellular membranes. Step 11 shows membrane repair processes, regulated by
dysferlin. Modified from Montecucco et al., 2008.
The release of the extracellular danger signal molecule ATP from muscle cells is
due to the alteration of the permeability of the sarcolemma, and occurs after a few
minutes of incubation with the toxins (Cintra-Francischinelli et al., 2010). ATP can
indirectly amplify the effect of muscle damage by binding to a variety of purinergic
receptors from the P2X family that allow transmembrane passage of K+, Na+, and Ca2+
(Cintra-Francischinelli et al., 2010). Moreover, the release of ATP, as well as the release of
K+, stimulates peripheral sensory neurons, which provides a basis for the pain sensation
after a snake bite (Cintra-Francischinelli et al., 2010). In this work, we have found that ATP
is released when myotoxins are injected in vivo in mice. However, when whole B. asper
venom was injected no significant release of ATP was detected. Since other viperid
venoms contain ATPase, ADPase and nucleotidase acivities, these activities were
29
quantified in the venom of B. asper and their role in venoms that contain myotoxins was
analyzed.
In order to shed light on some unknown aspects of the mechanism of action of
these myotoxins, a measurement of the hydrolysis of phospholipids caused by the
injection of the venom of B. asper, Mt-I, and Mt-II in Tibialis anterior muscles of CD-1 mice
was performed, as well as in cultured C2C12 myotubes exposed to both toxins. In ongoing
experiments, Mt-I is being labeled with a fluorophore and with biotin to study its
localization during its action on myotubes in culture and injected tibialis anterior muscles,
and also to isolate molecules that interact with the toxin.
30
Objectives
The first objective of my thesis work was that of testing the possibility that the
snake myotoxins Mt-I and Mt-II both act via the production of lysophospholipids and fatty
acids within the sarcolemma in C2C12 mouse myotubes in culture and in ex-vivo mouse
muscle using the quantitative methods offered by specialized mass spectrometers. This
indormation was essential to understand further the pathogenetic mechanism of action of
these important myotoxins.
The second objective was to quantify the ATPase, ADPase and nucleotidase
activities of B. asper venom, and to analyze their role in venoms that contain myotoxins.
The third objective was to conjugate enzymatically Mt-I with a fluorophore, in
order to study its localization in myotubes in culture and when injected in mice using
highly sensitive fluorescence imaging methods. A conjugation with biotin has also been
started, in order to isolate possible protein targets of Mt-I through a cross-linking reaction.
31
Chapter 1
Muscle phospholipid hydrolysis by Bothrops asper Asp49 and Lys49
phospholipase A₂ myotoxins - distinct mechanisms of action.
FEBS J. 2013;280(16): 3878-86.
32
33
34
35
36
37
38
39
40
41
42
43
Chapter 2. Why myotoxin-containing snake venoms possess powerful nucleotidases?
Biochem Biophys Res Commun. 2013; 430(4):1289-93.
44
45
46
47
48
49
Chapter 3.
Ongoing experiments
50
Introduction
Muscle damaging toxins are major components of the venom of B. asper, but
there is still a large gap in basic knowledge of their mechanism of action. It has been
hypothesized that these toxins first bind to acceptors in the plasma membrane of muscle
cells (Montecucco et al., 2008; Lomonte and Gutierrez; 2011). The identity of these
acceptors is still unknown. Moreover, the binding of the toxins and their localization
during their action on muscle cells has not been visualized, though in the first chapter of
this thesis results demonstrate that Mt-I has the ability to hydrolyze membrane
phospholipids of C2C12 myotubes in culture and of tibialis anterior muscles. The
transglutaminase of Streptomyces mobaraensis is being used to covalently modify Mt-I
with a fluorescent tag (ZQG-DNS fluorophore) and study its localization during its action
on muscle cells.
Transglutaminases are enzymes that form covalent bonds between the lateral
chains of lysine and glutamine. Therefore they can be used as a tool to modify proteins in
a reproducible way (Kamiya et al., 2009; Spolaore et al., 2012). Compared to chemically
labeled proteins, transglutaminase labeling results in defined label position(s) and degree
of labelling, minimized amounts of unlabelled protein, more solubility in water, and more
homogeneity (Kamiya et al., 2009; Spolaore et al., 2012).
Transglutaminases can be used to bind protein glutamines to molecules that
contain free amine groups or to bind protein lysines to glutamine containing peptides.
Generally, the residues modified by transglutaminases are located in flexible regions of
the protein (Spolaore et al., 2012). The Asp49 PLA2 Mt-I has been modified with a
glutamine containing peptide (ZQG, 1-N-Benzyloxocarbonyl-L-Glutaminyl-Glycinyl) with a
51
fluorescent tag (DNS, λEx max = 335 nm, λEm max = 550 nm). The localization of Mt-I
during its action on muscle cells is currently being studied. A biotin tag is also being added
to the Mt-I using the transglutaminase reaction, in order to determine which proteins
directly interact with the toxin.
52
Materials and methods
Venom and Mt-I
Venom of the snake B. asper was obtained from at least 40 specimens of the
Atlantic region of Costa Rica, kept at the serpentarium of Instituto Clodomiro Picado,
University of Costa Rica. Mt-I was purified from the venom based on the procedure of
Gutierrez et al. (1984), with further purification steps described in Fernandez et al. (2013).
Fluorescent Mt-I
To obtain the fluorescent Mt-I, a toxin solution (4.1 ml at a concentration of 1
mg/ml) dissolved in 0.1 M sodium phosphate buffer (pH=7) was added to 3.58 ml of 4 mM
1-N-(Benzyloxocarbonyl-L-Glutaminyl-Glycinyl)-5-N-(5´-N´,N´-dimethylamino-1´-naphtale-
nesulfonly) diaminopentane (Z-Gln-Gly-CAD-DNS, λEx max = 335 nm, λEm max = 550 nm,
ZEDIRA, Darmstadt, Germany) dissolved in 1 part of DMSO and 9 parts of 10 mM
hydrochloric acid. Then, 142 µl of a solution of transglutaminase from S. mobaraensis
(1.128 mg/ml) was added. After an incubation of 4 hours at 37 °C, the reaction was
stopped with 422 µl of 1 mM iodoacetamide. In order to purify the fraction of labeled Mt-I
from the un-labeled fraction, RP-HPLC was used, with the following conditions: a linear
gradient starting from 95 % of buffer A (water) and 5 % of buffer B (acetonitrile) to 30 %
buffer B in 3 minutes, followed by 30% of buffer B for 3 min, 30-50% B for 20 min and 50-
95% buffer B for 1 min. A flow of 1 ml/min and a C18 column (150 × 4.6 mm, 5 µm particle
size) was used. A control reaction without transglutaminase was carried out. A reaction in
which the ZQG peptide (without the fluorophore) was attached to Mt-I was also
performed.
53
Mass spectrometry
A Micromass (Manchester, U.K.) Q-Tof Micro mass spectrometer equipped with an
electrospray source (ESI-MS) was used for mass spectrometry analyses. Samples that
contained either Mt-I or fluorescent Mt-I were dissolved in 0.1% formic acid in an
acetonitrile/water mixture (1/1) and analyzed by MS. A capillary voltage of 3 kV, a cone
voltage of 35 V and an extractor voltage of 1 V were used in positive ion mode. For
Tandem MS (MS/MS) analyses, argon was used as collision gas. Masslynx 4.1 software
(Waters, Milford, MA, USA) was used for instrument control and data analysis.
To identify modification sites of the fluorescent Mt-I, 30 μg of the protein were
dissolved to a concentration of 1 mg/ml in 50 mM Tris-HCl pH 8.5, 5 mM TCEP and
incubated for 30 min at 37°C. Then, iodoacetamide 1 M was added at a final concentration
of 25 mM and the solution was further incubated in the dark for 20 min at room
temperature. The solution was acidified with an aqueous solution of 2 % trifluoroacetic
acid and the protein was purified by RP-HPLC using a linear gradient that started with 95 %
of buffer A (water) and 5 % of buffer B (acetonitrile) to 30 % buffer B in 3 minutes,
followed by 30% of buffer B for 3 min, 30-50% B for 20 min and 50-95% buffer B for 1 min.
Then the protein was lyophilized. Subsequently, it was dissolved in 0.1 M NH4HCO3 pH 8.0
at a concentration of about 1 mg/ml and incubated overnight at 37°C at an
enzyme/substrate ratio of 1/100 with trypsin. The reaction mixture was then diluted 1:2
with 0.1% formic acid in ACN: water (1:1) and then analysed by MS and MS/MS.
PLA2 activity assay
To measure PLA2 activity, the sPLA2 assay kit from Cayman Chemical (Cayman
Chemicals, Ann Arbor, MI, USA) was used. The 1,2-dithio of diheptanoyl
phosphatidylcholine analog was used as a substrate. After the hydrolysis of the thio-ester
54
bond, free thiols were detected using DTNB (5,5-dithio-bis-(2-nitrobenzoic acid)).
Absorbance was measured at 405 nm every minute after adding the substrate, to obtain
fifteen time points. Activity was expressed in µmol/min/ml.
Cells
Murine C2C12 skeletal muscle cells (CRL-1772) from the American Type Culture
Collection (Manassas, VA, USA) were used. Cells were maintained as myoblasts in
Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 10%
fetal bovine serum (EuroClone, Milan, Italy). 150.000 cells were plated in glass coverslips
(24 mm diameter) previously coated (overnight with 0.1 mg/ml poly-lysine, followed by
50 µg/ml collagen for 2 hours and subsequently 3 washes with phosphate buffered saline).
After 24 h, cells were differentiated into myotubes using Dulbecco’s modified Eagle’s
medium supplemented with 2% equine serum (Gibco) for 5–6 days. The medium was
changed every 24–48 h. Myotubes were then incubated with either Mt-I or fluorescent
Mt-I in a final volume of 500 µl modified Krebs–Ringer buffer (140 mM NaCl, 2.8 mM KCl,
2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES and 11 mM glucose, pH 7.4). In assays performed
in 96 well plates, 10000 C2C12 mioblasts per well were plated (volume per well: 100 µl).
Cells were first maintained in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum. After 1 day, cells were differentiated into myotubes using
Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum for 5–6 days.
The differentiation medium was replaced every 48 h.
Primary cultures of skeletal muscle cells were also used. Newborn (1–2 days old)
mice (CD1, Charles River) were used, as reported by Massimino et al. (2006). To obtain the
cells, posterior limb muscles were removed and washed in phosphate buffered saline
(PBS). The tissue was then minced and three successive treatments with trypsin (0.1% in
55
PBS, 30 min, 37 °C) were applied. Cells were resuspended in Ham’s F12 (Eurobio)
supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100
μg/ml streptomycin (Eurobio, proliferation medium). A quantity of 35000 cells was seeded
in 13 mm glass coverslips previously coated with collagen (0.1% in PBS) in 24 well plates.
After 48 h of incubation, the growth medium was replaced with Dulbecco-modified Eagle’s
medium (DMEM) containing 2% horse serum (Eurobio), to favour myoblast differentiation.
Cells were used the third day after differentiation.
Cytotoxicity assay
Assays were performed in 96 well plates. Cell vitality of C2C12 myotubes after
exposure to Mt-I or fluorescent Mt-I (25 µg/ml) dissolved in mKRB (final volume 100 μl)
was measured with the MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-
(4-sulfonphenyl)-2H-tetrazolium, inner salt) assay. The CellTiter 96 AQueous One Solution
Cell Proliferation Assay (Promega, Milan, Italy) was used, according to the instructions
from the manufacturer.
Mice
White male mice (25-30 g) were killed by cervical dislocation and immediately
tibialis anterior muscles were exposed and injected with 30 µg of fluorescent Mt-I
(dissolved in 10 mM HEPES, 150 mM NaCl and 50% glycerol, volume of injection: 22 µl) or
the same volume of the vehicle used to dissolve the toxins. A Hamilton syringe was used
for the injection. Muscles were excised and transferred to Eppendorf tubes containing 1
56
mL of physiological buffer (139 mM NaCl, 12 mM NaHCO3, 4 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 1 mM KH2PO4 and 11 mM glucose, pH 7.4) at 37 °C, and oxygenated with a gentle
flow of 95% O2 and 5% CO2. Muscle samples were incubated for 5, 15 and 30 min. Muscles
were then covered in OCT embedding matrix and frozen in liquid nitrogen. Sections of 10
µM were then prepared at -20 °C using a cryostat, placed in slides, mounted, and then
observed with fluorescence and confocal microscopes.
Confocal microscopy
A Leica SP5 confocal microscope was used to observe C2C12 myotubes, primary
myotubes and tibialis anterior muscles.
Ethics statement
All procedures involving animals were performed in accordance with the Italian
Animal Welfare Act (law 116/1992) and were approved by the local authority veterinary
service (project number 24088, 2011).
Results
57
Fluorescent Mt-I
The fluorescent Mt-I was purified and separated from Mt-I using RP-HPLC (Figure
7), after a transglutaminase reaction to attach the fluorescent peptide. Mass spectrometry
analysis showed that the fluorescent Mt-I had a molecular mass of 14605.06±0.58 Da
while Mt-I alone had a molecular mass of 13967.35±0.40 Da. The difference of 638 Da is
due to the addition of the fluorescent peptide ZQG-DNS (this peptide has a molar mass of
654.78 Da, and after the reaction ammonia is produced, therefore a difference of 638 Da
is obtained).
MS analysis of the triptic digestion of Mt-I-ZQG (without the fluorophore) indicated
that the glutamine containing peptide ZQG was added to lysine 105 of Mt-I (Figure 8).
Figure 7. Purification of fluorescent Mt-I by RP-HPLC. The run that contained unmodified
Mt-I is shown in blue, while the run that contained the fluorescent Mt-I is shown in black.
A linear gradient that started with 95 % of buffer A (water) and 5 % of buffer B
(acetonitrile) to 30 % buffer B in 3 minutes, followed by 30% of buffer B for 3 min, 30-50%
B for 20 min and 50-95% buffer B for 1 min was used. The difference in mass, measured
with a mass spectrometer, corresponds to the addition of the fluorescent peptide.
58
Figure 8. Localization of the conjugated ZQG-fluorophore in Mt-I. Tryptic digests of the
conjugated toxin were analyzed by Mass Spectrometry, as described in materials and
methods. Mt-I was labeled in lysine 105.
Cytotoxicity.
The fluorescent Mt-I retained its toxicity when in contact with C2C12
myotubes. There was however a loss in the percentage of toxicity when compared to the
unmodified Mt-I (Figure 9), due to the addition of the fluorescent peptide.
59
Figure 9. Comparison of the toxic activity of Mt-I and the fluorescent Mt-I in contact with
C2C12 myotubes. The vitality was measured after 45 minutes of incubation with the
toxins. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Milan,
Italy) was used, according to the instructions from the manufacturer. Error bars represent
the standard deviation of two independent experiments.
PLA2 activity
Addition of the glutamine containing peptide ZQG alone (without the fluorophore)
using the transglutaminase reaction did not affect the PLA2 activity of Mt I, since the PLA2
activity of the modified Mt-I was 94.1±16 µmol/min/mg and the unmodified Mt-I had an
activity of 93.7±11 µmol/min/mg. It will be tested in the future if the ZQG peptide with
the fluorophore also does not affect the PLA2 activity of Mt-I.
Fluorescent Mt-I in primary myotubes and ex vivo muscles
60
Fluorescent Mt-I rapidly binds to the membrane of myotubes in primary myotubes
and ex vivo tibialis anterior muscles (Figures 10 and 11), and the binding seems
discontinuous in both cases.
Figure 10. Visualization of Fluorescent Mt-I (25 µg/ml) in contact with primary mouse
myotubes. Cells were seeded and differentiated in 13 mm glass coverslips previously
coated with collagen. After incubation with the toxin, cells were fixated and viewed using
a confocal microscope.
61
Figure 11. Binding of fluorescent Mt-I to the membrane of tibialis anterior muscles. Mice
were killed, and immediately the tibialis anterior muscle was injected with 30 µg of
fluorescent Mt-I or with a control injection with the vehicle. Muscles were excised and
transferred to Eppendorf tubes containing 1 mL of physiological buffer, with a gentle flow
of oxygen. Muscles were incubated for the indicated periods of time, then covered in OCT,
frozen in liquid nitrogen and slides were prepared using a cryostat, as described in
materials and methods. Images were acquired with a confocal microscope.
62
Discussion
It has been inferred that the site of action of snake venom PLA2s is the
sarcolemma. However, the binding of these toxins to the plasma membrane has still not
been visualized. Transglutaminase labeling results in a defined label position and degree
of labelling, as well as good reproducibility of the labellling reaction. A very pure labelled
protein can also be obtained after its separation from the unlabelled protein by RP HPLC.
Therefore this approach was chosen to attach a DNS fluorophore to Mt-I. The fluorescent
Mt-I retained its toxicity as well as its PLA2 activity when it was modified enzymatically.
Mass spectrometry analysis of Mt-I ZQG (without the fluorophore) indicated that the
peptide was added to lysine 105 of Mt-I, where it does not interfere with the enzymatic
activity of the toxin.
Current results show that Mt-I rapidly binds to the membrane of myotubes, and
tibialis anterior muscles of mice. Future experiments will determine if Mt-I is able to enter
myotubes before cells die, or if the toxin acts exclusively from the membrane.
A biotin tag (ZQG-biotin) is also currently being attached to the Mt-I using the
transglutaminase reaction, to determine which proteins interact with the toxin, through a
crosslinking reaction.
63
Concluding remarks
PLA2s are abundant components in snake venoms, and exert an immense variety
of toxic and pharmacological effects. Considerable and important contributions have
been made to understand their structure and mechanism of action. However, many key
points in their mechanism of action still remain unsolved.
Both Mt-I and Mt-II from B. asper cause myonecrosis, but through different
mechanisms. This was confirmed in cell culture and tibialis anterior muscle in Chapter 1,
with a quantitative assessment of the species of phospholipids that were hydrolyzed. Mt-I
possesses PLA2 activity and hydrolyzes phospholipids while Mt-II acts through a
mechanism independent of PLA2 hydrolysis. Mass spectrometry analysis did not detect an
activation of the intracellular phospholipases in the case of cells and muscles in the
presence of Mt-II. Therefore, the mechanism of membrane damage of Mt-II does not
depend on the direct or indirect hydrolysis of phospholipids.
Mt-I hydrolyzed phospholipid species in a proportion that suggested that the main
site of action of the toxin is the external layer of the sarcolemma, in both cells in culture
and tibialis anterior muscles. Results obtained in chapter 3 show that the sarcolemma is
the first site of contact of Mt-I. The identity of the protein receptor, if any, still remains to
be deciphered, and is an immediate future goal that is being addressed with the
generation of a Mt-I covalently linked to Biotin. A cross-linking reaction, together with
mass spectrometry, would identify if there are proteins that interact with this toxin.
Even though hydrolytic enzymes with ATPase, ADPase and nucleotidase activities
are present in snake venoms from many species, the role of these proteins in the toxic
64
effects of snake venoms has not been the object of intense study. The findings that when
myotoxins where injected in vivo ATP was released, but when whole B. asper venom was
injected very low levels of released ATP were detected in the site of injection, indicated
that this effect could be due to the hydrolysis of released ATP. The confirmation of the
presence of these enzymes in the venom through the quantification of the ATPase,
ADPase and nucleotidase activities, gives an important toxic role to these proteins. These
hydrolytic enzymes act in combination with myotoxins and released ATP is converted to
adenosine, which is a multitoxin that has hypotensive, anticoagulant and paralyzing
activities.
65
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