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

Abstract

The venom of the black widow spider (BWSV) (Latro-dectus 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-latroinsectotoxin (-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids.-LIT comprises four structural domains: a signal pep-tide 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-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 (130 kDa) encoded in the-LIT gene differs from that of native-LIT purified from BWSV (110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native-LIT revealed a molecular ion with m/z of 110916 100, indicating that the native-LIT is 991 amino acids in length. When the full-length-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 neuro-transmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native-LIT have a high Ca 2 permeability, whereas those formed by truncated, recombinant protein do not.