| L.tredecimguttatus,also named black widow spider,is one of the most poisonous spiders found so far in the world.Different from other poisonous animals,L.tredecimguttatus has toxic components not only in its poison glands,but also in other parts of its body,such as the legs and abdomen,and even in the eggs as well as spiderlings.Now,new progresses have been achieved in the research on the spider egg toxins.Comprehensively utilizing physio-biochemical and proteomic techniques,our laboratory has made a systematic analysis of the molecular basis for the toxicity of L.tredecimguttatus eggs,finding that the eggs are rich in high-molecular-weight proteins and the egg toxicity is primarily caused by the proteinous components in the eggs;compared with the venom secreted by poisonous glands,the eggs have a more complex protein composition and there are only a few similarities in protein compositions of the two materials,suggesting that the eggs have a toxic mechanism different from that of the venom.Meanwhile,using mutiple biochemical separation techniques,systematic studies were made on the separation and purification of proteionous toxins from the eggs.Two novel toxic proteins,named Latroeggtoxin-I and Latroeggtoxin-?,respectively,were purified and characterized successively from the eggs.In orde to further investigate the active proteins in the L.tredecimguttatus eggs and probe into the molecular mechanisms for egg toxicity,the present study comprehensively employed gel filtration chromatography,ion exchange chromatography and reverse-phased high performance liquid chromatography to separate and purify the other proteins from the egg extract and then analyze the structure and properties of the purified protein.The crude extract of L.tredecimguttatus eggs was prepared by repeated grinding and extraction,and then fractionated by gel filtration chromatography into seven major peaks(named P1-P7).Preliminary toxicity experiments indicated that peak P1 contained the components with strong toxicities,and this fraction was therefore chosen to be further separated in the present study.When fraction P1 was further chromatographically separated on a DEAE ion exchange column,it was separated into five major peaks(named D1-D5).The components in major peak D4 were desalted and further separated with RP-HPLC on a C4 revrsed-phase column and finally a single protein component was acquired.SDS-PAGE analysis indicated that the protein sample was electrophoretically pure and the protein had a molecular weight of about 35 kDa.Toxicity analysis demonstrated that,at the experimental doses used,the purified protein had no obvious toxic effects on mouse,and the nerve-muscle transmission in isolated mouse phrenic nerve-diaphragm preparation as well as the sodium and potassium channel currents in rat dorsal root ganglion neurons were not significantly affected by the protein.However,the protein had strong toxic effects on P.americana.After being intraperitoneally injected with the protein at a dose of 10 ?g/g,the cockroaches displayed a series of obvious poisoning symptoms,including body turning,sluggishness and slow response,and finally died.When the possible effects of the protein on the ion channels in cockroach dorsal unpaired median neurons were investigated using the whole-cell patch-clamp technique,it was found that the protein had certain inhibitory effects on potassium and calcium channel currents.All the data suggest that the protein may be a kind of insect-specific toxin,which was named Latroeggtoxin-?.The N-termianl sequence of Latroeggtoxin-? was identified by Edman degradation to be S-T-K-S-S-E-S-L-Y-L-E-A-L-Y-I-D-K-M-T-H-E-P-V-A-D—.When this sequence was utilized to perform Sequence query and homology analysis with Mascot and BLAST softwares,respectively,no completely matched proteins were found,demonstrating that Latroeggtoxin-? is a novel toxic protein purified from the eggs of L.tredecimguttatus. |
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