Biochemical Research On Proteins From The Eggs Of The Spider L.tredecimguttatus

L.tredimguttatus,also named black widow spider,is a medium-sized spider,taxonomically belonging to Phylum Arthopoda,Arac:hnida,Araneae,Theridiidae,genus Latrodectus.lt is one of the most poisonous spiders found so far in the world.L.tredecimguttatus spiders have received increasing attention due to frequently reported human injures caused by them.A number of early studies and our previous work have systematiclly described the properties of the venom secreted by their poison glands and the structures and functions of some venomous proteins.Interestingly,L.tredecimguttatus spiders,different from other poisonous animals,have toxic components not only in the poison glands,but also in other parts of the body(such as legs and abdomen)and their eggs as well as spiderlings.Although the eggs of L.tredecimguttatus spiders have been demonstrated to be rich in biologically active components that exhibit well research value and application foreground,up to now there have been only sporadic reports on the related researches,lacking a systemnatic study on the toxicity of the eggs and the complete purification and structure-function analysis of the active proteins in the eggs.In the present study,an egg extract of L.tredecimguttatus spiders was prepared by repeated homogenization and extraction with a buffer of weak ionic strength or ddH20.After the extract was separated into two fractions(greater than or less than 10 kDa)with a 10 kDa ultrafilter,the two fractions were separately detected with mouse isolated phrenic nerve-hemidiaphragm preparations.The results indicated that the high-molecular-weight fraction in the egg extract exhibited strong toxicity.For investigating the possible relationship of proteins in the eggs with the toxic effect,the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spider's venom.SDS-PAGE showed that the proteins of the eggs were primarily distributed in the MW range of higher than 55 kDa as well as around 34 kDa.Totals of 157 proteins were identified from the egg extract using CapLC-MS/MS-based strategies.Bioinformatic abalysis indicated that these identified proteins were involved in multiple important cellular functions and metabolic processes,including catalysis,transport and metabolism regulation.Comparison indicated that the protein composition of the eggs was more complex than that of the venom and there were only a few similarities between the protein compositions of the two materials.Moreover,the study had not identified the known typical L.tredecimguttatus spider venomous proteins,latrotoxins,and other known venomous protenous components from the egg extract.These data demonstrate that the eggs have their own toxic mechanism different from that of the venom.In order to in more detail investigate the active proteins in the eggs of L.tredecimguttatus spiders,multiple physiological and biochemical techaniques were comprehensively employed to isolate the proteins from the eggs and preliminarily study the structure and function of the purified proteins.A neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography.Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry.Intraperitoneal injection of the protein in mice made the animals display a series of poinsoning symptoms.Electrophysiological experiments showed that the protein could block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner,demonstrating that the protein had strong mammal toxicity.The N-terminal sequence of the protein was identified by Edman degradation to be NSIAD DRYRW PGYPG AGLIP YIIDS_.When the sequence was used to search against protein database with Sequence Query in Mascot engine,there was no completely matched sequence or protein,while the BLAST analysis indicated that no significant similarity was found.These results demonstrate that the protein(named Latroeggtoxin-I)is a novel toxic protein purified from the eggs of L.tredecimguttatus spiders.Another novel proteinous toxin,named Latroeggtoxin-II,was purified from the eggs of L.tredecimguttatus spiders using a combination of gel filtration,ion exchange chromatography and reversed-phase chromatography.Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa,and its N-terminal sequence was determined by Edman degradation to be ESIQT STYVP NTPNQ KFDYE VGKDY-.By employing multiple proteomic strategies,the isolated protein was demonstrated to have only a few similarities to the existing proteins in the databases,suggesting that it was a novel toxic protein isolated from the eggs of L.tredecimguttatus spiders.After being abdominally injected into mice and P.americana,the protein could make the animals especially P.americana display a series of poisoning symptoms.Electrophysiological experiments demonstrated that the protein could selectively inhibit TTX-R Na+ channel currents in rat dorsal root ganglion neurons,showing a certain application prospect in the study of neurophysiology and the development of pharmaceuticals as well as insecticides.In view of the fact that biological membranes especially the plasma membrane are the important subcellular structures involving multiple cellular functions,our present study also made a preliminary analysis of the proteins in the shell membranes of the eggs using proteomic techaniues,and performed a study on the relavant methodology.The insoluble pellet left after preparation of the egg extract was extensively washed with ddH2O and the proteins in the memebranes were extracted with a detergent solution.The proteins in the mixture were separated by SDS-PAGE,digested with trypsin and analyzed using CapLC-MS/MS.After de-redundance,totals of 26 proteins were identified.Most of these proteins were predicted proteins or the proteins of unknown function,with a few with catalytic actiivty or other functions.It is speculated that the composition and function of the proteins in egg shell membranes have their own particularities,which needs further investigation.At present,membrane protein analysis has lagged behind that of soluble proteins.One of the main reasons is that most membrane proteins especially those integral to or tightly associated with lipid membranes are usually of high hydrophobicity,which adversely affects the solubilization,proteolysis and identification of the proteins.In this study,we present a novel polyvinylidene difluoride(PVDF)membrane-mediated sample preparation method for micro-scale membrane proteome analysis,using a rat liver cell membrane-enriched fraction as model material.The proteins in the fraction were extracted with a solution containing about 2%sodium dodecyl sulfate(SDS)and the protein solution was applied on a piece of PVDF membrane activated in advance,followed by drying and repeated washing in order to remove SDS and other salts.Quantitative determination indicated that about 84%of the SDS in sample was removed and protein loss was much less than 10%.For digesting the proteins adsorbed on PVDF membrane,four methods were used and compared.The protein identification demonstrated that dimethyl formamide(DMF)-assisted digestion was most effective with regard to the identification of membrane proteins particularly the highly hydrophobic multi-transmembrane proteins.Above data demonstrate that PVDF membrane-aided sample cleanup combined with DMF-assisted digestion has potential in the micro-scale membrane proteome analysis.