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.
Kiyatkin
N I
NI
School of Biological and Molecular Sciences, Oxford Brookes University, England.
Kulikovskaya
I M
IM
Grishin
E V
EV
Beadle
D J
DJ
King
L A
LA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001740:Black Widow Spider; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009498:Neurotoxins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013111:Spider Venoms / Q000096:biosynthesis* / Q000633:toxicity; D018411:Spodoptera
D009498:Neurotoxins; D011994:Recombinant Proteins; D013111:Spider Venoms; C074805:latroinsectotoxin; C020763:alpha-latrotoxin