Biochemical Research On The Toxins From L.Tredecimguttatus Spiderlings

Latrodectus tredecimguttatus(also called black widow spider)is a medium-sized spider.Due to the strong toxicity of the venom secreted by its venomous glands,it is considered to be one of the most venomous spiders known in the world.Since very early time,the researchers have known that L.tredecimguttatus is a kind of unusual venomous animal,with toxins not only in its venomous glands,but also in other parts of its body,such as legs and abdomen,in its eggs,and even in the newborn spiderlings.Up to date,the toxicity and its molecular basis of the venom and eggs have been investigated relatively thoroughly.However,there have been only a few studies on the newborn spiderlings of L.tredecimgu t ta tus,although the spiderlings have been demonstrated to be rich in toxins and other biologically active components that are of interest as tools for neurophysiological researches or as potential lead molecules for the development of insecticides and pharmaceuticals.In the present work,we performed a systematical analysis of the aqueous extract of newborn spiderlings of L.tredecimguttatus collected from inside the egg sacs stored at-20 ? in an attempt to reveal their toxicity and molecular basis.The extract of spiderlings was prepared using repeated homogenization-centrifugation.The extract was shown to contain 69.42%of proteins varying in molecular weights and pIs.Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms and death occurred when large doses were injected,with LD50 being 5.30 mg/kg in mice and 16.74 ?Pg/g in P.americana.Electrophysiological experiments indicated that the extract at a concentration of 10 ?g/mL could completely block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations within 21 ± 1.5 min.When applied at different concentrations,the extract inhibited HeLa cell proliferation in a dose-dependent manner,with an IC50 value of 158 ?g/mL.Flow cytometry indicated that treatment of HeLa cells with the extract induced apoptosis in HeLa cells in a dose-dependent manner,and led to cell cycle arrest in S phase.Enzymatic analysis indicated that application of the extract to the cultured HeLa cells increased the activity of caspase 3,a key apoptotic proteas,in the cells in a dose-dependent manner.Under the electron microscope,part of the HeLa cells treated with different concentrations of the extract appeared to be morphologically changed,displaying plasma membrane blebbing,nuclear fragmentation and condensation of chromatin,etc.Besides,patch clamp experiments demonstrated that 100 ?g/mL extract could inhibit the voltage-activated Na+,K+ and Ca2+ channel currents in rat DRG neurons to different extent,without affecting their ion selectivity and activation kinetics.These results demonstrated that the newborn spiderlings are rich in biologically active components and suggest a promising resource for the development of tool reagents and drugs such as anti-cancer agent.For obtaining even more detailed information on the biologically active components in the newborn spiderlings,the total extract was fractionated and the biological activity of the resulting fractions were separately detected.The extract was first fractionated with a 10000-dalton ultrafilter into high-molecular-mass fraction(>10 kDa)and low-molecular-mass fraction(<10 kDa).It was experimentally demonstrated that the low-molecular-mass fraction could not block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations,did not cause obvious morphological changes in HeLa cells,and had no significant effect on the HeLa cell proliferation,which suggested that the toxicity of the spiderling extract was primarily attributed to its high-molecular-mass components.In consideration of the important roles of high-molecular-mass fraction of the extract,we made further separation and activity analysis of the subfractions.The proteins in the fraction were further fractionated into four primary subfractions based on their sizes using gel filtration chromatography,named P1 to P4,all of which were found to inhibit the proliferation of HeLa cells.Using RP-HPLC with a C4 reversed-phase column,an active protein with a molecular mass of about 23 kDa,named PP23 protein,was purified from the subfraction P3.Analysis of biological activities showed that the PP23 protein could effectively inhibit HeLa cell proliferation,increase caspase 3 activity,and arrest HeLa cell in G2/M phase.These data indicate that the PP23 protein has cytotoxic and apoptotic activities against HeLa cells and the protein could inhibit the voltage-activated Na+currents in nude rat myocardial cell,thus suggesting a promising 'lead molecule for anti-cervical cancer and treating heart disease agent development.In view of the fact that both of the newborn spiderlings and adult spiders contain toxic components,we made a proteomic analysis in order to get a comparative insight into the molecular bases for their toxicities.SDS-PAGE indicated that the adult spider extract showed even more protein bands than spiderling extract.The high-abundance proteins of the adult spider extract were mainly distributed in about 30 kDa-70 kDa,while those of the spiderling extract in about 60 kDa-130 kDa.After in-gel digestion and analysis by CapLC-MS/MS,a total of 184 and 316 proteins were identified in the spiderling extact and adult spider extract.Of the identified proteins,140(76.87%)were identified only from the spiderling extract,272(86.08%)only from adult spider extract,and 44 from both of the two extracts,indicating that the protein compositions of spiderlings and adult spiders are quite different.Functional classification indicated that,among the proteins identified only from the spiderling extract,proteins with binding proteins accunted for 44%,followed by hydrolase(27%),others(18%),other enzymes(6%)and unknown proteins(5%),whereas the percentages of the corresponding proteins identified only from the adult spider extract were 44%,14%,13%,21%and 8%,respectively.These data indicate that there are significant differences between the protein of newborn spiderlings and adult spiders,the protein composition of the adult spiders is even more complex,and the L.tredecimguttatus spiderling has distinct molecular basis and mechanism for its toxicity.