Latrotoxin

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A latrotoxin is a high-molecular mass neurotoxin found in the venom of spiders of the genus Latrodectus (widow spiders) as well as at least one species of another genus in the same family, Steatoda nobilis . [1] Latrotoxins are the main active components of the venom and are responsible for the symptoms of latrodectism.

Contents

The following latrotoxins have been described: five insecticidal toxins, termed α, β, γ, δ and ε-latroinsectotoxins, one vertebrate-specific neurotoxin, alpha-latrotoxin, and one toxin affecting crustaceans, α-latrocrustatoxin. [2]

α-Latrotoxin

The best-studied latrotoxin is alpha-latrotoxin, which acts presynaptically to release neurotransmitters (including acetylcholine) from sensory and motor neurons, as well as on endocrine cells (to release insulin, for example). [3] It is a ~130 kDa protein that exists mainly in its dimerized or tetramerized forms.

α-Latrotoxin (α-LTX) can naturally be found in widow spiders of the genus Latrodectus . The most widely known of those spiders are the black widows, Latrodectus mactans . [4] The venom of widow spiders (Latrodectus) contains several protein toxins, called latrotoxins, which selectively target either vertebrates, insects or crustaceans. 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. [5]

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. [6]

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), 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. [6]

α-LTX precursor molecules are synthesized by free ribosomes in the cytosol and are therefore cytosolic in the secretory epithelial cells of the venom glands., [6] [7] 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 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. [8]

The α-LTX protein tertiary structure can be divided in three parts: the N-terminal wing (36 kDa), [7] the body (76 kDa), [7] and the C-terminal head (18.5 kDa). [7] 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. [8] Tetramer formation activates toxicity. [7]

Toxicokinetics

α-LTX affects motor nerve endings and endocrine cells. No major enzymatic activities are associated. [7] Instead, the toxin can form pores in the lipid membranes and induce Ca2+ 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. [7] [9]

Stimulation of small end-plate action potentials 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. [10]

Toxicodynamics

α-LTX in its tetrameric form interacts with receptors (neurexins and latrophilins) on the neuronal membrane, which causes insertion of α-LTX into the membrane.

Once the tetramer is inserted into the cell membrane, two mechanisms of action 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. [8] The four heads of the tetramer form a bowl surrounding the pore, which is restricted at one point to 10 Å. [7] Millimolar concentrations of Ca2+ and Mg2+ 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 La3+ higher than 100 μM also block tetramerisation. [7] 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). [7] 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. [7] 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 bi-layers is impossible when latrodectin is unavailable. Lactrodectin has no effect on α-LTX toxicity. [7]

Pore formation

The pores formed by α-LTX in the membrane are permeable to Ca2+ and therefore allow an influx of Ca2+ 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 Ca2+ strongly facilitates the forming of the tetramers and so its pore formation. The pore is also permeable to neurotransmitters, which causes massive leakage of the neurotransmitter pool in the cytosol. [8]

Alongside the influx of Ca2+, the channel is not very selective, allowing Na+, K+, Ba2+, Sr2+, Mg2+, 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 Yb3+, Gd3+, Y3+, La3+ and Al3+. [7]

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 [ citation needed ], 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. [ citation needed ]

Receptors

The following mechanism is suggested for receptor-mediated effects. Three receptors for α-latrotoxin have been described:

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 Ca2+ from intracellular stores. This rise in cytosolic Ca2+ may increase the probability of release and the rate of spontaneous exocytosis. [8] 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. [11]

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). [ citation needed ]

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 Ca2+ or Mg2+) 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. [7] [8]

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 Ca2+, whereas latrophilin and PTPσ can mediate insertion without presence of Ca2+. [8] 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. [8]

Toxicity

The median lethal dose (LD50) of α-LTX in mice is 20–40 μg/kg of body weight. [8]

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. [12]

Scientific contribution

αLTX has helped confirm the vesicular transport hypothesis of transmitter release, establish the requirement of Ca2+ 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. [8]

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 Ca2+ 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. [8]

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. [13]

High-molecular-weight proteins that have been isolated from the Mediterranean black widow (L. tredecimguttatus) include the insect-specific neurotoxins α-latroinsectotoxin and δ-latroinsectotoxin, a neurotoxin affecting crustaceans known as latrocrustatoxin, and small peptides that inhibit angiotensin-1-converting enzyme. [2]

Apart from the high molecular weight latrotoxins described above, Latrodectus venom also contains low molecular weight proteins [14] whose function has not been explored fully yet, but may be involved in facilitating membrane insertion of latrotoxins. [15]

Related Research Articles

<span class="mw-page-title-main">Exocytosis</span> Active transport and bulk transport in which a cell transports molecules out of the cell

Exocytosis is a form of active transport and bulk transport in which a cell transports molecules out of the cell. As an active transport mechanism, exocytosis requires the use of energy to transport material. Exocytosis and its counterpart, endocytosis, are used by all cells because most chemical substances important to them are large polar molecules that cannot pass through the hydrophobic portion of the cell membrane by passive means. Exocytosis is the process by which a large amount of molecules are released; thus it is a form of bulk transport. Exocytosis occurs via secretory portals at the cell plasma membrane called porosomes. Porosomes are permanent cup-shaped lipoprotein structure at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release intra-vesicular contents from the cell.

<span class="mw-page-title-main">Neuromuscular junction</span> Junction between the axon of a motor neuron and a muscle fiber

A neuromuscular junction is a chemical synapse between a motor neuron and a muscle fiber.

<span class="mw-page-title-main">Synaptic vesicle</span> Neurotransmitters that are released at the synapse

In a neuron, synaptic vesicles store various neurotransmitters that are released at the synapse. The release is regulated by a voltage-dependent calcium channel. Vesicles are essential for propagating nerve impulses between neurons and are constantly recreated by the cell. The area in the axon that holds groups of vesicles is an axon terminal or "terminal bouton". Up to 130 vesicles can be released per bouton over a ten-minute period of stimulation at 0.2 Hz. In the visual cortex of the human brain, synaptic vesicles have an average diameter of 39.5 nanometers (nm) with a standard deviation of 5.1 nm.

<span class="mw-page-title-main">End-plate potential</span> Voltages associated with muscle fibre

End plate potentials (EPPs) are the voltages which cause depolarization of skeletal muscle fibers caused by neurotransmitters binding to the postsynaptic membrane in the neuromuscular junction. They are called "end plates" because the postsynaptic terminals of muscle fibers have a large, saucer-like appearance. When an action potential reaches the axon terminal of a motor neuron, vesicles carrying neurotransmitters are exocytosed and the contents are released into the neuromuscular junction. These neurotransmitters bind to receptors on the postsynaptic membrane and lead to its depolarization. In the absence of an action potential, acetylcholine vesicles spontaneously leak into the neuromuscular junction and cause very small depolarizations in the postsynaptic membrane. This small response (~0.4mV) is called a miniature end plate potential (MEPP) and is generated by one acetylcholine-containing vesicle. It represents the smallest possible depolarization which can be induced in a muscle.

<span class="mw-page-title-main">SNARE protein</span> Protein family

SNARE proteins – "SNAPREceptors" – are a large protein family consisting of at least 24 members in yeasts, more than 60 members in mammalian cells, and some numbers in plants. The primary role of SNARE proteins is to mediate the fusion of vesicles with the target membrane; this notably mediates exocytosis, but can also mediate the fusion of vesicles with membrane-bound compartments. The best studied SNAREs are those that mediate the release of synaptic vesicles containing neurotransmitters in neurons. These neuronal SNAREs are the targets of the neurotoxins responsible for botulism and tetanus produced by certain bacteria.

<span class="mw-page-title-main">Latrodectism</span> Medical condition

Latrodectism is the illness caused by the bite of Latrodectus spiders. Pain, muscle rigidity, vomiting, and sweating are the symptoms of latrodectism.

α-Bungarotoxin Chemical compound

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Cytolysin refers to the substance secreted by microorganisms, plants or animals that is specifically toxic to individual cells, in many cases causing their dissolution through lysis. Cytolysins that have a specific action for certain cells are named accordingly. For instance, the cytolysins responsible for the destruction of red blood cells, thereby liberating hemoglobins, are named hemolysins, and so on. Cytolysins may be involved in immunity as well as in venoms.

<span class="mw-page-title-main">Emodepside</span> Chemical compound

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<span class="mw-page-title-main">Neurexin</span> Protein family

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<span class="mw-page-title-main">Latrophilin 1</span> Protein-coding gene in the species Homo sapiens

Latrophilin 1 is a protein that in humans is encoded by the ADGRL1 gene. It is a member of the adhesion-GPCR family of receptors. Family members are characterized by an extended extracellular region with a variable number of protein domains coupled to a TM7 domain via a domain known as the GPCR-Autoproteolysis INducing (GAIN) domain.

<span class="mw-page-title-main">Latrophilin 3</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Latrophilin 2</span> Protein-coding gene in the species Homo sapiens

Latrophilin 2 is a protein that in humans is encoded by the ADGRL2 gene.

<span class="mw-page-title-main">KCNB1</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Active zone</span>

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Edwin R. Chapman is an American biochemist known for his work on Ca2+-triggered exocytosis. He currently serves as the Ricardo Miledi Professor of Neuroscience at the University of Wisconsin–Madison, where he is also an investigator of the Howard Hughes Medical Institute (HHMI).

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References

  1. ↑ Dunbar, John P.; Fort, Antoine; Redureau, Damien; Sulpice, Ronan; Dugon, Michel M.; Quinton, Loïc (June 2020). "Venomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis". Toxins. 12 (6): 402. doi: 10.3390/toxins12060402 . PMC   7354476 . PMID   32570718.
  2. 1 2 Grishin EV (November 1998). "Black widow spider toxins: the present and the future". Toxicon. 36 (11): 1693–701. doi:10.1016/S0041-0101(98)00162-7. PMID   9792186.
  3. ↑ Südhof TC (2001). "alpha-Latrotoxin and its receptors: neurexins and CIRL/latrophilins". Annu. Rev. Neurosci. 24: 933–62. doi:10.1146/annurev.neuro.24.1.933. PMID   11520923. S2CID   906456.
  4. ↑ Südhof, TC (2001). "alpha-Latrotoxin and its receptors: neurexins and CIRL/latrophilins". Annual Review of Neuroscience. 24: 933–62. doi:10.1146/annurev.neuro.24.1.933. PMID   11520923. S2CID   906456.
  5. ↑ Ushkaryov, YA; Volynski, KE; Ashton, AC (April 2004). "The multiple actions of black widow spider toxins and their selective use in neurosecretion studies". Toxicon. 43 (5): 527–42. doi:10.1016/j.toxicon.2004.02.008. PMID   15066411.
  6. 1 2 3 Ushkaryov, YA; Volynski, KE; Ashton, AC (April 2004). "The multiple actions of black widow spider toxins and their selective use in neurosecretion studies". Toxicon. 43 (5): 527–42. doi:10.1016/j.toxicon.2004.02.008. PMID   15066411.
  7. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ushkaryov, YA; Rohou, A; Sugita, S (2008). alpha-Latrotoxin and its receptors. Handbook of Experimental Pharmacology. Vol. 184. pp. 171–206. doi:10.1007/978-3-540-74805-2_7. ISBN   978-3-540-74804-5. PMC   2519134 . PMID   18064415.
  8. 1 2 3 4 5 6 7 8 9 10 11 Ushkaryov, YA; Volynski, KE; Ashton, AC (April 2004). "The multiple actions of black widow spider toxins and their selective use in neurosecretion studies". Toxicon. 43 (5): 527–42. doi:10.1016/j.toxicon.2004.02.008. PMID   15066411.
  9. ↑ Henkel, AW; Sankaranarayanan, S (May 1999). "Mechanisms of alpha-latrotoxin action". Cell and Tissue Research. 296 (2): 229–33. doi:10.1007/s004410051284. PMID   10382267. S2CID   9933831.
  10. ↑ Peterson, ME (November 2006). "Black widow spider envenomation". Clinical Techniques in Small Animal Practice. 21 (4): 187–90. doi:10.1053/j.ctsap.2006.10.003. PMID   17265903.
  11. ↑ Hiramatsu, H; Tadokoro, S; Nakanishi, M; Hirashima, N (December 2010). "Latrotoxin-induced exocytosis in mast cells transfected with latrophilin". Toxicon. 56 (8): 1372–80. doi:10.1016/j.toxicon.2010.08.002. PMID   20708026.
  12. ↑ Jelinek, GA (November 1997). "Widow spider envenomation (latrodectism): a worldwide problem". Wilderness & Environmental Medicine. 8 (4): 226–31. doi: 10.1580/1080-6032(1997)008[0226:WSELAW]2.3.CO;2 . PMID   11990169.
  13. ↑ Rohou A, Nield J, Ushkaryov YA (March 2007). "Insecticidal toxins from black widow spider venom". Toxicon. 49 (4): 531–49. doi:10.1016/j.toxicon.2006.11.021. PMC   2517654 . PMID   17210168.
  14. ↑ Gasparini S, Kiyatkin N, Drevet P, et al. (August 1994). "The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones". J. Biol. Chem. 269 (31): 19803–9. doi: 10.1016/S0021-9258(17)32091-4 . PMID   8051061.
  15. ↑ Graudins, Andis; Little, Michelle J.; Pineda, Sandy S.; Hains, Peter G.; King, Glenn F.; Broady, Kevin W.; Nicholson, Graham M. (1 January 2012). "Cloning and Activity of a Novel α-latrotoxin from Red-back Spider Venom". Biochemical Pharmacology. 83 (1): 170–183. doi:10.1016/j.bcp.2011.09.024. hdl: 10453/18571 . PMID   22001442.
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