A latrotoxin is a high-
molecular mass
The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The related quanti ...
neurotoxin
Neurotoxins are toxins that are destructive to nerve tissue (causing neurotoxicity). Neurotoxins are an extensive class of exogenous chemical neurological insultsSpencer 2000 that can adversely affect function in both developing and mature ner ...
found in the venom of
spider
Spiders ( order Araneae) are air-breathing arthropods that have eight legs, chelicerae with fangs generally able to inject venom, and spinnerets that extrude silk. They are the largest order of arachnids and rank seventh in total species ...
s of the genus ''
Latrodectus
''Latrodectus'' is a broadly distributed genus of spiders with several species that are commonly known as the true widows. This group is composed of those often loosely called black widow spiders, brown widow spiders, and similar spiders. Howeve ...
'' (widow spiders) as well as at least one species of another genus in the same family, ''
Steatoda nobilis''. Latrotoxins are the main active components of the venom and are responsible for the symptoms of
latrodectism
Latrodectism () is the illness caused by the bite of ''Latrodectus'' spiders (the black widow spider and related species). Pain, muscle rigidity, vomiting, and sweating are the symptoms of latrodectism.
There are several spider species all named ...
.
The following latrotoxins have been described: five
insecticidal toxins, termed α, β, γ, δ and ε-latroinsectotoxins, one vertebrate-specific
neurotoxin
Neurotoxins are toxins that are destructive to nerve tissue (causing neurotoxicity). Neurotoxins are an extensive class of exogenous chemical neurological insultsSpencer 2000 that can adversely affect function in both developing and mature ner ...
, alpha-latrotoxin, and one toxin affecting
crustacean
Crustaceans (Crustacea, ) form a large, diverse arthropod taxon which includes such animals as decapods, seed shrimp, branchiopods, fish lice, krill, remipedes, isopods, barnacles, copepods, amphipods and mantis shrimp. The crustacean group ...
s, α-latrocrustatoxin.
α-Latrotoxin
The best-studied latrotoxin is alpha-latrotoxin, which acts presynaptically to release
neurotransmitter
A neurotransmitter is a signaling molecule secreted by a neuron to affect another cell across a synapse. The cell receiving the signal, any main body part or target cell, may be another neuron, but could also be a gland or muscle cell.
Neuro ...
s (including
acetylcholine
Acetylcholine (ACh) is an organic chemical that functions in the brain and body of many types of animals (including humans) as a neurotransmitter. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Part ...
) from sensory and motor neurons, as well as on endocrine cells (to release
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
, for example). It is a ~130 kDa
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
that exists mainly in its dimerized or tetramerized forms.
α-Latrotoxin (α-LTX) can naturally be found in widow spiders of the genus ''
Latrodectus
''Latrodectus'' is a broadly distributed genus of spiders with several species that are commonly known as the true widows. This group is composed of those often loosely called black widow spiders, brown widow spiders, and similar spiders. Howeve ...
''. The most widely known of those spiders are the black widows, ''
Latrodectus mactans
''Latrodectus mactans'', known as southern black widow or simply black widow, and the shoe-button spider, is a venomous species of spider in the genus ''Latrodectus''. The females are well known for their distinctive black and red coloring and f ...
''. The venom of widow spiders (''Latrodectus'') contains several protein toxins, called latrotoxins, which selectively target against either
vertebrate
Vertebrates () comprise all animal taxa within the subphylum Vertebrata () ( chordates with backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the phylum Chordata, ...
s,
insect
Insects (from Latin ') are pancrustacean hexapod invertebrates of the class Insecta. They are the largest group within the arthropod phylum. Insects have a chitinous exoskeleton, a three-part body ( head, thorax and abdomen), three pairs ...
s or
crustacean
Crustaceans (Crustacea, ) form a large, diverse arthropod taxon which includes such animals as decapods, seed shrimp, branchiopods, fish lice, krill, remipedes, isopods, barnacles, copepods, amphipods and mantis shrimp. The crustacean group ...
s. One of these toxins is α-latrotoxin and targets selectively against vertebrates; it is ineffective in insects and crustaceans. α-LTX has a high affinity for receptors that are specific for neuronal and endocrine cells of vertebrates.
Biosynthesis
As the DNA sequence for α-LTX is transcribed and translated, an inactive precursor molecule of α-LTX (156.9 kDa) is formed. This precursor molecule undergoes post-translational processing where the eventual, active α-LTX protein (131.5 kDa) is formed.
The N-terminus of the α-LTX precursor molecule is preceded by short hydrophilic sequences ending with a cluster of basic amino acids. These clusters are recognized by proteolytic enzymes (furin-like
proteases
A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the for ...
), which cleave and activate the α-LTX precursor molecules by means of hydrolysis. The C-terminus too is recognized by these furin-like proteases and is also cleaved.
α-LTX precursor molecules are synthesized by free
ribosomes
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to f ...
in the
cytosol
The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells (intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...
and are therefore cytosolic in the secretory
epithelial
Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...
cells of the venom glands.,
They can, however, associate with secretory granules although they are not taken up in the lumen of the granules. The cytosolic α-LTX precursor molecule is released from the cell by means of
holocrine
Holocrine (from Ancient Greek ὅλος; ''hólos'', “whole, entire” + κρῑ́νω; ''krī́nō'', “to separate”) is a term used to classify the mode of secretion in exocrine glands in the study of histology. Holocrine secretions are p ...
secretion where it ends up in the venom gland of the spider. This gland contains the several proteases involved in the cleavage of the precursor α-LTX molecule.
The α-LTX protein tertiary structure can be divided in three parts: the N-terminal wing (36 kDa),
the body (76 kDa),
and the C-terminal head (18.5 kDa).
Because of C-terminal ankyrin repeats, which mediate protein-protein interactions, the α-LTX monomer forms a dimer with another α-LTX monomer under normal conditions.
Tetramer formation activates toxicity.
Toxicokinetics
α-LTX affects motor nerve endings and endocrine cells. No major enzymatic activities are associated.
Instead, the toxin can form pores in the lipid membranes and induce Ca
2+ ion flow.
The onset of effects by intoxication can occur with a lag-period of 1 to 10 minutes, even at subnanomolar concentration levels. At nanomolar concentrations, bursts of neurotransmitter release occur. After the bursts, prolonged periods of steady-state release take effect.
Stimulation of small
end-plate action potentials
An action potential occurs when the membrane potential of a specific cell location rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells, c ...
are initially induced by the neurotoxin, while later on the neurotransmission is blocked at the neuromuscular junction. This is due to depletion of synaptic vesicle contents.
Toxicodynamics
α-LTX in its tetrameric form interacts with receptors (
neurexin
Neurexins (NRXN) are a family of presynaptic cell adhesion proteins that have roles in connecting neurons at the synapse. They are located mostly on the presynaptic membrane and contain a single transmembrane domain. The extracellular domain ...
s and
latrophilin
Latrophilins are a group of highly conserved G-protein coupled receptors from the adhesion G protein-coupled receptor family. These receptors were originally identified based on their ability to bind to a component of black widow spider venom ...
s) on the neuronal membrane, which causes insertion of α-LTX into the membrane.
Once the tetramer is inserted into the cell membrane, two mechanism of actions can occur. First, insertion may lead to pore formation and possibly other effects, and second, the receptor may be activated, which leads to intracellular signaling.
The four heads of the tetramer form a bowl surrounding the pore, which is restricted at one point to 10 Å.
Millimolar concentrations of Ca
2+ and Mg
2+ strongly catalyze tetramer formation, suggesting that the tetrametric state is divalent cation-dependent, while EDTA favours formation of the dimer. Research also shows that concentrations of La
3+ higher than 100 μM also block tetramerisation.
Pore formation can occur in pure lipid membranes, but reconstituted receptors greatly increase pore formation. Biological membranes block pore formation when no α-LTX receptors are present (neurexin, latrophilin, PTPσ).
It is also known that the three highly conserved cysteine residues are involved with α-LTX receptor binding, because mutants containing serine instead of cysteine residues did not induce toxicity.
The N-terminal domain needs to fold properly, in which the disulfide bonds need to be functional. The α-LTX toxin is bound by a small protein, LMWP or latrodectin. It has been observed that pore formation in lipid bilayers is impossible when latrodectin is unavailable. Lactrodectin has no effect on α-LTX toxicity.
Pore formation
The pores formed by α-LTX in the membrane are permeable to Ca
2+ and therefore allow an influx of Ca
2+ into the cell. This influx into an excitable cell stimulates exocytosis directly and efficiently. The cation influx is proportional to the amount of pores and hence the amount of involved receptors expressed on the cell membrane. Also Ca
2+ strongly facilitates the forming of the
tetramers
A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula Ti ...
and so its pore formation. The pore is also permeable to neurotransmitters, which causes massive leakage of the neurotransmitter pool in the
cytosol
The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells (intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...
.
Alongside the influx of Ca
2+, the channel is not very selective, allowing Na
+, K
+, Ba
2+, Sr
2+, Mg
2+, Li
+ and Cs
+ to pass the membrane too. The pore is open most of the time, with an open probability of 0.8. Most trivalent cations block channels at 50-100 μM, such as Yb
3+, Gd
3+, Y
3+, La
3+ and Al
3+.
The pore is not only permeable for cations, but also for water. This causes nerve terminal swelling. Further membrane potential disturbances occur due to permeability of small molecules, such as neurotransmitters and ATP to pass through the α-LTX pore.
;Membrane penetration
Although tetrameric pore formation of α-latrotoxin has been shown conclusively , some authors still dispute whether this is the main mode of action of α-latrotoxin, and believe that α-latrotoxin (tetrameric or not) may penetrate through the membrane of target cells to interact directly with intracellular neurotransmitter release machinery.
Receptors
The following mechanism is suggested for receptor-mediated effects.
Three receptors for α-latrotoxin have been described:
*
neurexin
Neurexins (NRXN) are a family of presynaptic cell adhesion proteins that have roles in connecting neurons at the synapse. They are located mostly on the presynaptic membrane and contain a single transmembrane domain. The extracellular domain ...
*
latrophilin
Latrophilins are a group of highly conserved G-protein coupled receptors from the adhesion G protein-coupled receptor family. These receptors were originally identified based on their ability to bind to a component of black widow spider venom ...
(aka CIRL, Calcium-Independent Receptor for Latrophilin)
*
protein tyrosine phosphatase sigma
Protein tyrosine phosphatases (EC 3.1.3.48, systematic name protein-tyrosine-phosphate phosphohydrolase) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins:
: proteintyrosine phosphate + H2O = ...
(PTPσ).
The toxin stimulates a receptor, most likely latrophilin, which is a G-protein coupled receptor linked to Gαq/11. The downstream effector of Gαq/11 is phospholipase C (PLC).When activated PLC increases the cytosolic concentration of IP3, which in turn induces release of Ca
2+ from intracellular stores. This rise in cytosolic Ca
2+ may increase the probability of release and the rate of spontaneous exocytosis.
Latrophilin with α-LTX can induce the activation of Protein Kinase C (PKC). PKC is responsible for the phosphorylation of SNARE proteins. Thus latrophilin with α-LTX induces the effect of exocytosis of transport vesicles. The exact mechanism has to be discovered.
;Signaling
As well as the major effects of α-latrotoxin pore formation, other effects of α-latrotoxin are mediated by interaction with latrophilin and intracellular signalling (see
signal transduction
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation catalyzed by protein kinases, which ultimately results in a cellula ...
).
Structure activity relationship (SAR)
The natural occurring α-LTX dimer has to form a tetramer to be toxic. Tetramerisation occurs only in the presence of bivalent cations (such as Ca
2+ or Mg
2+) or amphipathic molecules. The four monomers that form this tetramer are symmetrically arranged around a central axis, resembling a four-blade propeller with a diameter of 250 Å and a thickness of 100 Å. The head domains form the compact, central mass brought together and surrounded by the body domains. The wings stand perpendicular towards the axis of the tetramer. Because of this form the tetramer contains a pear-shaped channel in the central mass. At the lower end the diameter of this channel is 25 Å, then widens to 36 Å to be constricted to 10 Å at the top.
The base of the tetramer (below the wings) is 45 Å deep and is hydrophobic, which mediates insertion into the cell membrane. Also insertion of the tetramer is only possible in presence of certain receptors (mainly neurexin Iα and latrophilin and PTPσ in a minor extent) on the membrane. Neurexin Iα only mediates insertion under presence of Ca
2+, whereas latrophilin and PTPσ can mediate insertion without presence of Ca
2+.
So because of the channel and the insertion in the cell membrane the protein makes the cell more permeable to substances that can pass through the channel. These substances are mono- and bivalent cations, neurotransmitters, fluorescent dyes and ATP.
Toxicity
The LD50 of α-LTX in mice is 20–40 μg/kg of body weight.
The LD50 of ''Latrodectus'' venom in mg/kg for various species: frog = 145, blackbird = 5.9, canary = 4.7, cockroach = 2.7, chick = 2.1, mouse = 0.9, housefly = 0.6, pigeon = 0.4, guinea-pig = 0.1.
Scientific contribution
αLTX has helped confirm the vesicular transport hypothesis of transmitter release, establish the requirement of Ca
2+ for vesicular exocytosis, and characterize individual transmitter release sites in the central nervous system. It helped identify two families of important neuronal cell-surface receptors.
The mutant form of αLTX, which is called αLTXN4C and does not form pores, has contributed to research. It helped the approach to deciphering the intracellular signaling transduction mechanism stimulated by αLTX. The mutant toxin can also be used to study the nature and properties of intracellular Ca
2+ stores implicated in the toxin receptor transduction pathway and their effect on evoked postsynaptic potentials. The mutant toxin can also be an instrument to elucidate the endogenous functions of αLTX.
Other venom components
The natural prey of widow spiders are insects, and several insectotoxins are found in its venom. The latroinsectotoxins appear to have similar structures.
High-molecular-weight proteins that have been isolated from the
Mediterranean black widow
''Latrodectus tredecimguttatus'', also known as the Mediterranean black widow, or the European black widow, is a species in the genus ''Latrodectus'' of the widow spiders. It is commonly found throughout the Mediterranean region, ranging from ...
(''L. tredecimguttatus'') include the insect-specific neurotoxins α-latroinsectotoxin and δ-latroinsectotoxin, a neurotoxin affecting crustaceans known as latrocrustatoxin, and small
peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A ...
s that inhibit
angiotensin-1-converting enzyme.
[
Apart from the high molecular weight latrotoxins described above, ''Latrodectus'' venom also contains low molecular weight proteins] whose function has not been explored fully yet, but may be involved in facilitating membrane insertion of latrotoxins.
References
{{Acetylcholine metabolism and transport modulators
Neurotoxins
Spider toxins