Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family: functional expression requires C-terminal truncation.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
Dulubova
I E
IE
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Krasnoperov
V G
VG
Khvotchev
M V
MV
Pluzhnikov
K A
KA
Volkova
T M
TM
Grishin
E V
EV
Vais
H
H
Bell
D R
DR
Usherwood
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry; D001483:Base Sequence; D001740:Black Widow Spider / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D007313:Insecta; D008051:Lipid Bilayers; D013058:Mass Spectrometry; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000187:drug effects / Q000502:physiology; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology
D017487:Ankyrins; D017931:DNA Primers; D018076:DNA, Complementary; D008051:Lipid Bilayers; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D013111:Spider Venoms; C098875:delta-latroinsectotoxin; C074805:latroinsectotoxin