The cloning of a cDNA encoding a protein (latrodectin) which co-purifies with the alpha-latrotoxin from the black widow spider Latrodectus tredecimguttatus (Theridiidae).
A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
Pescatori
M
M
Istituto di Biologia Cellulare, CNR, Roma, Italy.
Bradbury
A
A
Bouet
F
F
Gargano
N
N
Mastrogiacomo
A
A
Grasso
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001740:Black Widow Spider / Q000737:chemistry*; D003001:Cloning, Molecular; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D013111:Spider Venoms / Q000032:analysis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D007106:Immune Sera; D012333:RNA, Messenger; D013111:Spider Venoms; C094437:latrodectin; C020763:alpha-latrotoxin