FUNCTIONAL-CHARACTERIZATION OF BLACK-WIDOW SPIDER NEUROTOXINS SYNTHESIZED IN INSECT CELLS

alpha-latrotoxin, alpha-latroinsectotoxin and the low-molecular-mass p rotein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombin ant-virus-infected cells revealed novel proteins that migrated with si zes similar to those of the neurotoxins from spider venom. The identit ies of these proteins as alpha-latrotoxin, alpha-latroinsectotoxin or the low-molecular-mass protein were confirmed by immunoblot analysis o f infected cells with anti-(alpha-latrotoxin), anti-(alpha-latroinsect otoxin) or anti-(low-molecular-mass protein) IgG. Neither the low-mole cular-mass protein nor alpha-latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus-derived synthesis directly in th e cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding alpha-latroinsectotoxin however, rev ealed a strong toxic effect on the T. ni larvae. These data indicate t hat 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 inte rnalisation of part of the neurotoxin alpha-subunit molecule.