Functional Characterization of Black Widow Spider Neurotoxins Synthesised in Insect Cells

Abstract

α‐latrotoxin, α‐latroinsectotoxin and the low‐molecular‐mass protein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombinant‐virus‐infected cells revealed novel proteins that migrated with sizes similar to those of the neurotoxins from spider venom. The identities of these proteins as α‐latrotoxin, α‐latroinsectotoxin or the low‐molecular‐mass protein were confirmed by immunoblot analysis of infected cells with anti‐(α‐latrotoxin), anti‐(α‐latroinsectotoxin) or anti‐(low‐molecular‐mass protein) IgG. Neither the low‐molecular‐mass protein nor α‐latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus‐derived synthesis directly in the cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding α‐latroinsectotoxin, however, revealed a strong toxic effect on the T. ni larvae. These data indicate that the toxic effect of the native insectotoxin may be promoted by the α‐latroinsectotoxin subunit alone and provides evidence that the mechanism of action of α‐latroinsectotoxin may be mediated by internalisation of part of the neurotoxin α‐subunit molecule.

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

“…Given the abundance of latrodectins in black widow spider venom, and evidence of paralogs that experienced negative and positive selection, it is likely that latrodectins plays some key role in prey acquisition. However, past functional studies indicated that purified latrodectins are not toxic to mammals or insects (Kiyatkin et al, 1995; Volkova et al, 1995). For example, purified latrodectin 1 and 2 from L. tredecimguttatus is non-toxic to cockroaches ( Periplaneta americana ) when injected at doses of up to 80 μg/g, having not caused lethality or paralysis (Volkova et al, 1995).…”

“…Latrodectins are peptides of ~70 amino acids that cannot be separated from latrotoxins using standard protein purification (Kiyatkin et al, 1992, 1990; Pescatori et al, 1995; Volkova et al, 1995). Multiple studies have demonstrated that purified latrodectin is not toxic in insects and mammals (Gasparini et al, 1994; Grishin et al, 1993; Kiyatkin et al, 1995; Volkova et al, 1995). However, latrodectins may function as subunits of a latrotoxin complex (Kiyatkin et al, 1992), even though latrotoxins do not require latrodectins for neurotransmitter release (Dulubova et al, 1996; Grishin et al, 1993; Kiyatkin et al, 1995; Volynski et al, 1999).…”

Penelope’s web: using α‐latrotoxin to untangle the mysteries of exocytosis

“…2006). Consistent with its obligatory processing in the venom gland, the pro‐toxin is inactive (Kiyatkin et al. 1995).…”

“…1990) shows that α‐LTX is synthesised on free cytosolic ribosomes in the spider’s venom glands as a large pro‐toxin, which is proteolytically processed in the lumen of the venom gland to produce the mature toxin with a molecular mass of ∼130 kDa (see Ushkaryov 2002). The latter contains two domains: the N‐terminal one‐third of the molecule has no significant sequence homology to known proteins, while the C‐terminal two‐thirds contain 22 ankyrin‐like repeats (ALRs), the most prominent feature of α‐LTX (Kiyatkin et al. 1995).…”

“…Sequence analysis (Kiyatkin et al 1990) and immunocytochemistry (Cavalieri et al 1990) shows that a-LTX is synthesised on free cytosolic ribosomes in the spider's venom glands as a large pro-toxin, which is proteolytically processed in the lumen of the venom gland to produce the mature toxin with a molecular mass of 130 kDa (see Ushkaryov 2002). The latter contains two domains: the Nterminal one-third of the molecule has no significant sequence homology to known proteins, while the C-terminal two-thirds contain 22 ankyrin-like repeats (ALRs), the most prominent feature of a-LTX (Kiyatkin et al 1995). ALRs are found in a wide variety of unrelated proteins with diverse functions, and are thought to mediate intra-and/or intermolecular interactions (Li et al 2006).…”