Screening And Inhibitory Activity Of Polypeptide Toxin Acting On Type Ⅱ Transmembrane Serine Protease Matriptase

Due to the heterogeneity of tumors and the complexity of tumor microenvironment,the mechanism of many tumors occurrence has not been clearly understood.Matriptase is overexpressed in a variety of tumor tissues,and participates in the degradation of various protein substrates（such as Pro-HGF,Pro-UPA and PAR-2）in the cell membrane and extracellular matrix in vivo,and promotes the occurrence and development of tumors by enhancing the proliferation,infiltration and migration of tumor cells.Therefore,Matriptase,as the key nodes in the invasion and metastasis pathways of tumor cells,have always been considered as an important target of anti-tumor therapy.To screen peptides that target to inhibit Matriptase in a safe and efficient manner,and this study plans to filter active ingredients with Matriptase inhibition by using the animal venom resource library in the laboratory.In this study,the MT-SP1 domain of Matriptase（596-856 aa）was expressed and purified by escherichia coli expression system with PET24a vector.The enzyme activity of MT-SP1 was determined by enzyme kinetic parameters.By using MT-SP1 to catalyze the chromogenic reaction of Pefachrome TPA substrate,toxins with inhibitory effect on the activity of protease MT-SP1 were screened out from more than 10 species of venom,such as Artempeda argyroda,five-step snake,C hinese cobra,mesocornis and scorpion.The results showed that TPA degradation of MT-SP1 was significantly inhibited by the crude venom of TPA.The activity of protease was inhibited by 85%in 1mg/m L and 72%in0.1mg/m L.After RP-HPLC isolation and purification,the active components of C.armoides（the peak time,25.6 min）could significantly inhibit the degradation of TPA substrate by protease MT-SP1.The half inhibitory concentration（IC50）of MT-SP1 degradation substrate is 0.165μM,and the inhibitory parameter Ki value of MT-SP1 is 0.163μM by formula conversion.Using Edman degradation of the active component protein determination,N-terminal sequence alignment long frost spider c DNA library of predicting peptide sequence after sequence was confirmed as the following:ACGHLHDPCKPGPPSTN TCCIGLQCRYGSCLVQV（p I/Mw:7.83/3559.14）.The compound is named THTX-Cl19 based on the international standard naming rules for polypeptide toxins.And,the active peptide with the same molecular weight is separated and purified by the method of chemical synthesis and oxidation refolding.After detecting the effect of THTX-Cl19 on the degradation of TPA by MT-SP1,we further verified the effect of THTX-C l19 on the hydrolysis of macromolecular substrates Pro-HGF and Pro-UPA of MT-SP1.MT-SP1 is able to self-hydrolyze and activate recombinant mouse Pro-HGF substrate with His label attached to C-terminal,and then cleavage to matureα-HGF andβ-HGF.The C-terminal of cleavedβ-HGF still carries HIS label.Western Blot results show that THTX-Cl19 inhibits the degradation of pro-HGF substrate by MT-SP1 in a concentration-dependent and time-dependent manner.Similarly,the recombinant pro-u PA protein with His label attached to its C-terminal is also the cleavage substrate of MT-SP1,and the active A-u PA and B-u PA are generated by proteolysis.THTX-Cl19 also inhibits Matriptase cleavage of pro-u PA substrates in a concentration-and time-dependent manner.Protease selectivity assay shows that THTX-Cl19 effectively inhibits the enzyme activity of trypsin,and the Ki value was 0.371μM.However,different concentration of THTX-C l19 treatment almost has no significant effect on the degradation ability ofα-chymotrypsin and matrix metalloproteinase（MMP-2）.The selectivity experiments of voltage-gated ion channels show that the stimulation with 10μM THTX-Cl19weakly inhibits the current of Nav1.5,Kv3.1 and Kv3.2 ion channels（22.3%,6.5%and 13.9%,respectively）.However,there is no significant inhibition on the other Nav and K v ion channel currents detected.According to different sources,two invasive prostate cancer cell lines（PC-3 cell line with high expression of Matriptase,DU145 cell line with no expression of Matriptase）and a normal human prostate epithelial cell line RWPE-1 are selected in this study to test whether THTX-C l19 inhibit Matriptase-mediated carcinogenesis.The cytotoxic activity,anti-proliferation ability and anti-migration/invasion activity of THTX-Cl19 are determined by colony formation assay,CCK-8 assay,scratch healing assay and Transwell chamber migration/invasion assay.Cell proliferation assay shows that THTX-Cl19 effectively inhibits the proliferation of PC-3 prostate cancer cells,while the proliferation ability of DU145 and RWPE-1 cell lines is not affected.Anti-migration assay show that THTX-C l19 significantly inhibits cell migration in transwell compartment.Different concentrations of THTX-Cl19 do not affect the DU145 prostate cancer cells migration.And,there is almost no cell migration in the RWPE-1 cell line.The scratch healing experiment shows that THTX-Cl19 effectively slows down the scratch healing of PC-3 prostate cancer cells,while THTX-Cl19 ha S little effect on cell migration in DU145 cell line.In addition,transwell invasion assay using matrigel to simulate extracellular matrix in vitro show that THTX-Cl19significantly inhibits transwell compartment invasion in PC-3 prostate cancer cells.The results of transcriptome studies in PC-3 prostate cancer cell lines show that the THTX-Cl19 treatment can significantly affect the regulation of prostate cancer pathway and tumor-related signaling pathways such as erb B,TNF,NF-Kappa B,MAPK,PI3K-Akt,m TOR and HIF-1 signaling pathways.Treatment with THTX-Cl19 also significantly reduces the gene expressed level of HAI-2（an endogenous inhibitor of Matriptase）,and increases the expression level of PTEN（a key tumor suppressor gene in prostate cancer）,and C DKN1A（an important gene regulating cell cycle）.Using the high homologous protein templates 2LL1 and 1X5V,the spatial structure of THTX-C l19 is constructed.The C-terminal of the simulated structure has stableβ-folded andα-helical structures.The simulated structure is an ICK-motif polypeptide with three pairs of disulfide bonds,which is composed of C ys _I-Cys _IV,Cys _II-Cys _V and C ys _III-Cys _VI.The MT-SP1＿SFTI-1 complex chain A（PDB ID 3P8F）is used as the receptor protein for protein docking experiments,and the optimal transient binding conformation of THTX-Cl19 interacting with the protease active domain MT-SP1 is obtained.After molecular dynamics simulation,the non-bond interaction analysis on the binding surface of THTX-Cl19＿MT-SP1 complex shows that the ligand THTX-Cl19 mainly formed 16 pairs of intermolecular hydrogen bond interaction,5 pairs ofπinteraction and 1 pair of salt bridge with the S2-S4 pocket,60-loop loop and other regions of protease MT-SP1.During the binding process of the complex formed between THTX-Cl19 and the active site of the protease domain MT-SP1,the Van der Waals interaction contributes the most to the binding of MT-SP1＿THTX-C l19 complex.The decomposition data of the binding free energy of MT-SP1 residues in the protease catalytic domain shows that Trp212（-8.10 kcal/mol）,His42（-3.47 kcal/mol）,Arg48（-2.31 kcal/mol）and other residues contribute more to the free energy of the complex formation.In conclusion,an ICK-motif structural polypeptide THTX-C l19 with Matriptase inhibitory activity is screened from the toxin of Cyriopagopus longipes in this study.THTX-Cl19 can effectively inhibit the degradation of different substrates by MT-SP1in vitro（Ki=0.163μM）.THTX-Cl19 significantly inhibits the proliferation,migration and invasion of PC-3 prostate cancer cell lines with high expression of Matriptase.THTX-Cl19 has high selectivity to MT-SP1 protease.It has been found that THTX-Cl19 can inhibit trypsin reaction（K i=0.371μM）,but has no obvious inhibition effect on other proteases and voltage-gated ion channels.