Elsevier

Toxicon

Volume 43, Issue 5, April 2004, Pages 527-542
Toxicon

Review
The multiple actions of black widow spider toxins and their selective use in neurosecretion studies

https://doi.org/10.1016/j.toxicon.2004.02.008Get rights and content

Abstract

The black widow spider venom contains several large protein toxins—latrotoxins—that are selectively targeted against different classes of animals: vertebrates, insects, and crustaceans. These toxins are synthesised as large precursors that undergo proteolytic processing and activation in the lumen of the venom gland. The mature latrotoxins demonstrate strong functional structure conservation and contain multiple ankyrin repeats, which mediate toxin oligomerisation. The three-dimensional structure has been determined for α-latrotoxin (αLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of αLTX pore formation by showing that it forms tetrameric complexes, harbouring a central channel, and that it is able to insert into lipid membranes. All latrotoxins cause massive release of neurotransmitters from nerve terminals of respective animals after binding to specific neuronal receptors. A G protein-coupled receptor latrophilin and a single-transmembrane receptor neurexin have been identified as major high-affinity receptors for αLTX. Latrotoxins act by several Ca2+-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant αLTX that does not form pores has been used to dissect the multiple actions of this toxin. As a result, important insights have been gained into the receptor signalling and the role of intracellular Ca2+ stores in the effect of αLTX.

Introduction

The notorious black widow spider (genus Latrodectus) belongs to the family of comb-footed cobweb spiders (Theridiidae: Arthropoda, Arachnida, Araneae) (Platnick, 1993, Platnick, 1997). Black widows are cosmopolitan and found throughout much of the world: China, Central Asia (L. mactans tredecimguttatus and L. lugubris, or karakurt), southern Europe (L. mactans tredecimguttatus), North and South America (L. mactans, L. geometricus, L. hesperus), India and Australia (L. hasselti, or red-back spider). The venom of Latrodectus spp. is a powerful stimulant of neurosecretion in different classes of animals (Longenecker et al., 1970, Frontali et al., 1972, Griffiths and Smyth, 1973, Kawai et al., 1972).

In addition to Latrodectus, the Theridiidae family includes more than 50 other genera; two of these, Steatoda and Achaearanea, are closely related to Latrodectus and, being particularly synanthropic, are routinely misidentified as black widows. The symptoms of Steatoda spp. envenomation resemble those of latrodectism (Warrell et al., 1991) and can be treated successfully with red-back spider antivenom (South et al., 1998, Graudins et al., 2002). However, antibodies against the principal component of the black widow venom, α-latrotoxin (αLTX), do not cross-react with Steatoda venom (Cavalieri et al., 1987), suggesting that some other functional constituents of these theridiid venoms are similar (see also Gillingwater et al., 1999).

Over the last thirty years, the toxins from the black widow spider venom—latrotoxins—have been extensively used to study the molecular mechanisms of neurosecretion in vertebrates, insects and crustaceans. Recent advances in the structural and functional analysis of some latrotoxins, especially αLTX, have radically improved our understanding of the complex actions of these toxins and brought about important insights into the mechanisms regulating neurotransmitter release. This review will summarise the contemporary views regarding the structures and modes of action of latrotoxins and their application as neurobiology tools.

Section snippets

The specialisations of latrotoxins

Most of the work on the biochemistry and physiology of latrotoxins was carried out using venoms from the Mediterranean and Central Asian varieties of L. mactans.

The first attempt to separate the venom from L. mactans tredecimguttatus, using gel-filtration followed by ion-exchange chromatography, without achieving complete purity, clearly indicated the presence of several proteins selectively toxic to vertebrates, insects or crustaceans (Granata et al., 1972, Frontali et al., 1976, Knipper et

Primary structures

To date, four latrotoxins have been cloned, using cDNA or intron-less genomic DNA from L. mactans tredecimguttatus, and sequenced: αLTX, αLIT, δLIT, and αLCT (Kiyatkin et al., 1990, Kiyatkin et al., 1993, Dulubova et al., 1996, Volynskii et al., 1999, Danilevich and Grishin, 2000). Surprisingly, the molecular masses of all these toxins deduced from their DNA sequences are substantially higher than those determined by SDS electrophoresis (Grasso, 1976, Krasnoperov et al., 1990a, Krasnoperov et

Pore formation

αLTX has long been known to form Ca2+-permeable pores in the plasma membrane of cells sensitive to it (Grasso et al., 1980). However, only with the determination of its 3D structure has it become possible to explain how the hydrophilic αLTX is able to insert itself into the membrane (Orlova et al., 2000). Furthermore, the understanding of the mechanism of pore formation has helped to identify its specific effects on secretion and to study the receptor-mediated αLTX actions in isolation.

The

αLTX receptors

Latrotoxins trigger exocytosis only after binding to neuronal receptors, and the identification of such receptors is likely to reveal the important mechanisms of presynaptic regulation of neurotransmitter release. At present, only αLTX receptors have been identified and characterised.

Surprisingly, several structurally and functionally unrelated cell-surface receptors for αLTX have been found. The first receptor to be discovered was neurexin Iα (NRX) (Ushkaryov et al., 1992), a neuronal protein

Modes of action of latrotoxins

The vertebrate-specific αLTX has been studied in detail, and its mode of action will be discussed below with reference to the other latrotoxins. Much of the early work on the action of black widow spider venom and its constituent toxins has been extensively discussed in the previous reviews (e.g. Rosenthal and Meldolesi, 1989), and we will concentrate here on the major advances made over the last decade. Although the actions of αLTX are complex, they can be divided into two major pathways:

Latrotoxins-versatile tools to study neuroexocytosis

All known latrotoxins have been used to some extent to analyse the mechanisms involved in the regulation of neurotransmitter release, although most of our knowledge comes from the application of αLTX. Thus, among other important findings, this toxin has helped to: confirm the vesicular hypothesis of transmitter release (Ceccarelli and Hurlbut, 1980, Hurlbut et al., 1990), establish the requirement of Ca2+ for endocytosis (Ceccarelli and Hurlbut, 1980), characterise individual transmitter

Acknowledgements

We thank all other (present and past) members of this laboratory and our collaborators for making important contributions to the structural and functional studies of latrotoxins. We thank A. Rohou for preparing the stereo-images of αLTX. The work is supported by a Wellcome Senior European Research Fellowship and a BBSRC project grant (to Y. A. U.)

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