Functional Characterization of Black Widow Spider Neurotoxins Synthesised in Insect Cells

Abstract

alpha-latrotoxin, alpha-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 alpha-latrotoxin, alpha-latroinsectotoxin or the low-molecular-mass protein were confirmed by immunoblot analysis of infected cells with anti-(alpha-latrotoxin), anti-(alpha-latroinsectotoxin) or anti-(low-molecular-mass protein) IgG. Neither the low-molecular-mass protein nor alpha-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 alpha-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 alpha-latroinsectotoxin subunit alone and provides evidence that the mechanism of action of alpha-latroinsectotoxin may be mediated by internalisation of part of the neurotoxin alpha-subunit molecule.

Molecular Evolution of α-Latrotoxin, the Exceptionally Potent Vertebrate Neurotoxin in Black Widow Spider Venom

“…Although their general role in stimulating neurotransmitter secretion is also similar, experimental evidence suggests the latrotoxins vary in target specificity. For example, α-latroinsectotoxin and δ-latroinsectotoxin appear to selectively affect insect neurons, whereas α-latrocrustotoxin stimulates secretion from crustacean neurons, but not from certain insects (Fritz et al 1980; Krasnopernov et al 1991; Magazanik et al 1992; Kiyatkin et al 1995; Elrick and Charlton 1999). The fourth paralog, α-latrotoxin, is a vertebrate-specific neurotoxin that causes the extreme pain following black widow bites (Knipper et al 1986; Kiyatkin et al 1990; Volynski, Nosyreva, et al 1999).…”

Cloning and Structure of δ-Latroinsectotoxin, a Novel Insect-specific Member of the Latrotoxin Family

“…The proteins are polypeptides of more than 1300 amino acids and share 34% amino acid identity. Although ␣-LTX and ␣-LIT have been expressed in a recombinant baculovirus expression system (11), it is not clear whether the full-length proteins are toxic. Also, when ␣-LTX was purified from BWSV, it was tightly associated with an acidic low molecular weight protein (12,13).…”

Poneratoxin, a neurotoxin from ant venom

“…The two helices have quite different characters. The first, PLLILGS (3)(4)(5)(6)(7)(8)(9), is apolar, whereas the second, IQ-AIHDAQR (17)(18)(19)(20)(21)(22)(23)(24)(25), contains polar and charged amino acids. This will result in different interactions with cell membranes.…”

“…It seems likely that when not exported, this peptide is either destroyed inside the cell or is toxic for the cells. Sequence analysis suggested that the part LPLLILGSLLMTPPVIQA (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), which looks like a transmembrane segment is similar to signal peptides of a variety of proteins [34]. If without an authentic signal peptide this fragment is seen by the expressing cell as a signal peptide, it could be degraded by the appropriate enzymatic system such as one that cleaves the signal peptide of preprolactine within its hydrophobic core, between two leucine clusters [35].…”

“…To overcome this limitation, several attempts have been made to obtain baculoviruses with enhanced toxicity. Recombinant baculoviruses have been constructed with genes coding for regulators of insect metabolism such as hormones and enzymes [2,3], but also for natural toxins of scorpions, mites, or spiders [4][5][6][7][8]. When compared with the wild-type virus, some of these recombinants were able to reduce the life span of infected insects.…”