| Tarantulas belong to the arthropoda,phyllometa,arachnida,and arachnids,which are widely distributed in the world,they are ranked fourth in the arachnids according to species diversity.In recent years,Thrombin is an important target for the development of procoagulant and anticoagulant drugs,ASIC3 channel is an important target for the treatment of pain caused by extracellular acidification,Screening of modulators of Thrombin and ASIC3 is still a current research focus.In a previous study,a novel peptide toxin LCTX-F2 was isolated and identified from the venom of Lycosa singoriensis with activity to promote coagulation and inhibitory effect on acid-sensitive ion channel.In terms of coagulation factors,This toxin can increase their protease activities of coagulation factors FXa,FXIIa,Thrombin and Kallikrein,and promote blood coagulation.In terms of acid-sensitive channels,LCTX-F2 reversibly inhibits ASIC1 a and ASIC3 channel currents,with IC50 of 5.8 μM and 4.6 μM,respectively.However,the key sites of LCTX-F2 in promoting coagulation factor activity and inhibiting acid channel activity are unknown.The peptide toxin LCTX-F2 contains 65 amino acid residues,consisting of 4 disulfide bonds forming a stable ICK(inhibitor cysteine knot)peptide motif structure.NCBI-BLAST sequence alignment shows that LCTX-F2 has high homology with multiple sequences in the transcriptome of cave tarantula(Lycosa singoriensis),but the function of these homologous polypeptides is unknown.Therefore,LCTX-F2 will have reference value for the study of homologous polypeptides in the future.According to the properties of the polypeptide,16 mutants(K2E,R7 E,R15E,R20 E,D10A,D10 R,D14A,D14 R,D26A,D26 R,F31A,V41 A,G35A,S43 A,E54A,and K61A)were successfully constructed on the LCTX-F2 prokaryotic expression vector by site-directed mutation technology.The pure polypeptide was obtained by prokaryotic expression technology and reversed-phase liquid chromatography.The changes of LCTX-F2 mutant on Thrombin and ASIC3 activity were further studied.In the detection of LCTX-F2 mutation in increasing Thrombin protease activities,when the positively charged amino acids at positions 2,7,and 15 were mutated to negatively charged amino acids(K2E,R7 E,R15E),the activity decreased by 42.4%,64.5 %,44.6%,especially the replacement of arginine at position 7 with glutamate significantly reduced the activity of LCTX-F2.The negatively charged amino acid at position 10 is mutated to the positively charged amino acid(D10R),and the activity is reduced 24.5%.However,amino acid mutations in other positions contributed little to the binding of LCTX-F2 to Thrombin.Because of two C-terminal mutants of the 16 designed mutants had no significant difference in activity,it was speculated that multiple amino acids of 2、7、10 and 15 at the N-terminus of LCTX-F2 played an important role in the influence of prothrombin activity.On the other hand,in the r ASIC3 inhibitory activity test of LCTX-F2 mutant,the inhibitory activities of the arginine at position 7 and aspartic at position 10(R7E,D10 A,D10R)were significantly weakened,and only inhibited approximately 27.3%,18.1%,and 8.3% of r ASIC3 currents compared with the control at Long-term stimulation of 50 μM R7 E,D10A,and D10 R,respectively,especially the mutation of lysine at position 2 to glutamic acid(K2E),which the inhibitory activity and selectivity of r ASIC3 is significantly enhanced Compared with LCTX-F2,the IC50 up to 0.35 μM.For other parts amino acid mutations had no obvious effection,indicating that K2、R7 and D10 is a key amino acid residue relative to the r ASIC3 channel bioactivity of LCTX-F2.In summary,in the study of the key site of the peptide toxin LCTXF2 to promote coagulation factor activity and inhibit Acid-sensing ion channel activity,we found that the N-terminus of LCTX-F2 is the key site of activity,especially the key residue arginine at position 7 significantly reduced peptide activity,and provided a basis for the subsequent analysis of the structure-function relationship of LCTX-F2 with coagulation factors and ASIC3 channels.At the same time,the engineered mutant K2 E is an inhibitor of ASIC3 channel with higher activity and selectivity,which can be used as a new tool molecule to contributes to the study of the structure and function of ASIC3 and drug lead molecules in the treatment of diseases caused by extracellular acidification. |
