Functional characterization of black widow spider neurotoxins synthesised in insect cells.

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.

Cloning and structure of cDNA encoding alpha-latrotoxin from black widow spider venom.

cDNA encoding the putative alpha-latrotoxin precursor was isolated from spider venom glands cDNA library and sequenced. The cDNA contained the 4203 base-pair open reading frame corresponding to the 156,855-Da protein composed of 1401 amino acids. Computer analysis of the deduced primary structure revealed the presence of various internal imperfect repeats mainly in its central and C-terminal regions.