Mediator Toxinomics Research: Focus On The Serine Protease Family

Snake venom contains many useful components.Recent technological advances in mass spectrometry and next generation sequencing have greatly simplified both proteomic and transcriptomic studies of snake venoms.Transcriptomes of snake venom glands are now routinely sequenced on a variety of platforms,allowing the examination of more components than that from traditional melthods.High-throughput profiling of snake venom glands allowed us to find transcriptions of the genes from mRNA level.In particular,Illumina sequencing,has allowed more accurate quantification of mRNA composition.However,in addition to venom proteins,next generation cDNA sequencing also detects many non-venom components.The advent of LC/MS-based venom proteomics permits high throughput screening of venom toxins.This approach relies on existing databases of protein sequences,and can be limited by the availability of reference data while mass spectrometry can validate cDNA sequencing.Combined use of next generation cDNA sequencing and LC/MS have considerable power to explore the snake venom proteins.Gloudius intermedius is the most toxic species among six pit-vipers in China,which subordinates to the Gloudius,Crotalinae of Viperidae,and distributes in northwest and northern China.With the goal of understanding their venom composition and the biological functions,here both techniques(next generation cDNA sequencing and LC-MS/MS)were used to explore the venoms of Gloudius intermedius.The main methods and results were briefly summarized as follows:1.Transcriptome of the venom gland of Gloydius intermedius(1)A total of 19821 unigenes from the venom gland cDNA library were sequenced by platform of Illumina Hiseq 2500,BlastX search revealed that 10547(53.21%)unigenes were annotated in the NR databases,among them,27 unigenes belonged to toxin-families.The five most abundant toxin families among the toxin unigenes were;metalloproteinase(MP,6),phospholipase A2(PLA2,6),C-type lectin like protein(CTLP,4),serine protease(SP,3),three-finger toxins(3FTxs,3).While only 1 unigene for hyaluronidase,phosphodiesterase,cysteine-rich secretory protein,L-amino acid oxidase and nerve growth factor,respectively.(2)The statistical analysis based on RPKM suggested that the toxin expressison levels(follow the decreasing order)were as followed:PLA2(82435.79)>SP(30758.48)>CTLP(6562.87)>L-AAO(6347.89)>NGF(4857.31)>CRISP(1204.57)>MP(1020.96)>PDE(191.29)>Hydase(148.39)>3FTx(27.93).(3)Phylogenetic tree of pitviper svSPs with newly identified nine Gisps isoforms shows that Gisp1 and Gisp3 are Kallikrein type SP;Gisp2 is serum C protein activator type SP;Gisp4 and Gisp5 are Cogulating type SP;Gi-PA1-4 are Plasminogen activators.(4)Relative expression level evaluated by quantitative PCR(qPCR)revealed that the Gisp expressison levels followed a decreasing order of Gisp1>Gi-PA1>Gisp2>Gi-PA2>Gisp3>Gi-PA3>Gisp4>Gi-PA4>Gisp5.2.Proteome of the Gloydius intermedius venomFrom the LC-MS/MS results,it revealed that totally 1146 specific peptides have been identified for nine toxin families,ie 510,356,192,27,26,12,12,7,2,2 specific peptides for SP,LAAO,PLA2,MP,CTLP,CRISP,BPP-CNP,PDE,Hydase and NFG,respectively.It is worth noting that no partial peptides have been identified for PLB and 3-FTx.Comparison of venom gland transriptome and proteome results,we found that SP,LAAO and PLA2 were the most highly expressed three toxin families.3.Purification and activity determination of Serine proteases from the venom of Gloydius intermediusSerine protease family showed the highest diversities,which have potential utilization value in clinical treatment for cardiovascular disease.Based on the results from transcriptome and proteome analysis,here,both Size exclusion chromatography(SEC)and anion exchange chromatography(AEC)were adopted to purify Gisp from the crude venom,then four specific chromogenic substances were used to determine the biological activities and distribution of different Gisp isoforms in different fractions,whereas SDS-PAGE was used to analyze protein purity,the main procedures and results were as followed:(1)Six fractions(PI-6)were obtained from SEC(Sephacryl-200HR)of G.intermedius crude venom.Using BApNA as the substrate,we found that serine protease activities mainly distributed in P3 fraction,SDS-PAGE analysis showed that P3 is not single band therefore need further step purification.(2)Secondly,fraction P3 was separated with AEC(on a HiTrap DEAE-FF 16/60 column)and five fractions were obtained(P3-1?P3-5).Using N-Benzoyl-Phe-Val-Arg-p-nitroanilide(B7632),N-?-tosyl-Gly-pro-Arg-p-Nitroanilide(T1637),H-D-Pro-Phe-Arg-nitroanilide(B2133),H-D-Val-Leu-Lys-p-nitroanilide(V7127)as substrates to determine the biological activities of five fractions above-mentioned.The results showed that kallikrein activities of all the five fractions(P3-1?P3-5)are relatively low;P3-1 and P3-2 displayed higher thrombin activities and thrombin-like activities,while fibrinolytic activities and kallikrein-like activities are relatively low;P3-3 displayed higher fibrinolytic activity and thrombin activity.Surprisingly,judging by the position ranging from 26 to 35 kDa(serine protease)of the five fractions,we found that P3-1and P3-2 held a high content of serine protease,but displayed low serine protease activity,while P3-3 was opposite.P3-4 and P3-5 displayed low hydrolytic activities of all the four substrates,indicating that P3-4 and P3-5 fractions barely contained serine proteases.In combination with the results of prediction of physical and chemical properties of the nine Gisps,we postulated that Gisp3,Gisp4 and Gisp5 mainly distributed in fraction P3-1,Gispl mainly distributed in fraction P3-2,while Gi-PA1,Gi-PA2 and Gi-PA3 mainly distributed in P3-3,Gi-PA4 distributed in fractions P3-1or P3-2.Conclusion:(1)Among the Gloydius pitvipers only G.intermedius evolves with unique neurotoxic venom,while other Gloydius species or populations contain typical hemorrhagic venom.Like those observed in some rattlesnakes of the New World,the present study highlights the venom bimorphism among the Gloydius pitvipers.Furthermore,our results demonstrate a trend of co-evolution of hypotensive components(BPP-CNP and kininogenase type SP)in certain viperid venoms.(2)Only one P-? MP but not P-? or P-? MPs(&disintergrin)are detected in G.i.venom.Nine Gisps isoforms are expressed,Gispl and Gisp3 are Kallikrein type;Gisp2 is C-protein activator;Gisp4 and Gisp5 are Cogulating enzymes;Gi-PA1-4 are Plasminogen activators.(3)Except CRISP and hyaluronidase,most of the venom toxins of G.intermedius are structurally highly(>90%)similar to those of other Gloydius or rattlesnakes.Rapid venom evolution should occur primarily through changes in the proportions of key toxins and new type of toxin isoforms,rather than point mutations affecting coding sequences.Similar conclusion has been reached in a recent study on C.horridus venomics.