Study On The Structure And Bioactivity Of Serine Protease Inhibitors From Venoms Of Two Myriapoda Species

Centipedes(Chilopoda)and Millipedes(Diplopoda)belong to the subphylum Myriapoda,phyla Arthropoda.They are widely distributed around the world.Centipedes and millipedes are live in a dark and humid environment,and equipped with unique defensive/venom glands to facilitate their survival or predation during long term struggles with natural enemies or predators.There are large quantity of active peptides with high specificity and functional diversity in their venoms.Centipedes and millipedes have been recorded in Chinese traditional medicine that can be used to cure many diseases with high efficiency.With the advancement of various research methods and technologies,active molecular components of millipedes and centipedes have been developed.Peptides with a variety of functional activities were isolated from the venom of centipedes,and they have rich activities such as antibacterial,antiplatelet aggregation,inhibition of ion channels,and proteases components.A large number of small organic molecules have been isolated from millipedes' defensive glands,which can be used as industrial catalysts,but there are no reports of peptide components in secretions of defensive glands.Proteases are ubiquitous in all organisms and can participate in a series of physiological processes.Depending on the catalytic mechanism,a variety of proteases have been identified.The presence of protease inhibitors can balance protease activity and provide the basis for normal physiological activities.Serine protease inhibitors are the largest group among them.Serine protease inhibitors of arthropods can act on the host's immune system or coagulation system,providing conditions for their predation;or prevent degradation of proteases secreted by other pathogens,providing conditions for their survival.Arthropod-derived natural serine protease inhibitors have been explored extensively.The two species,centipedes and millipedes,have hundreds of millions of years of evolutionary history of struggling with harsh environments.However,there are few studies on the activity of serine protease inhibitors in them.In order to understand whether serine protease inhibitors exist in centipedes and millipedes,peptides with inhibitory activity on serine protease were screened in Scolopendra hainanum and Prospirobolus joannsi.Two peptides were identified,and their functions were well studied.In the first part of this article,a peptide with 34 AA was identified in S.hainanum,which named ShSPI and with a sequence of CPQVCPAIYQPVFDEFGRMYSNSCEMQRARCLR G.The sequence comparison results showed that ShSPI belongs to the Kazal family and has a conserved site of protease inhibitors.Linear ShSPI was obtained by chemical synthesis and ShSPI with natural structure was refolded successfully by oxidizing the internal disulfide bonds of linear ShSPI.In the enzyme activity experiment in vitro,the Ki values of ShSPI on PPE and HNE were 225.83±20 nM and 12.61±2 nM,respectively.The dissociation constants of ShSPI on PPE and HNE were 1.0×10-7 M and 4.2×10-8 M,respectively.ShSPI have no effect on the activity of trypsin,chymotrypsin,FXa,FXIIa,plasmin,cathepsin G,thrombin and kallikrein.In conclusion,ShSPI exists strong and specific activity against elastase.The disulfide bonds pairing of Cysl-Cys31 and Cys5-Cys24 in ShSPI was determined by partial reduction and stepwise sequence analysis.The three-dimensional structure of ShSPI was analysed through NMR experiments.The results showed that ShSPI is differ from other Kazal domain and belongs to the atypical Kazal family.ShSPI has only one ?-helix and two reverse ?-sheets,which form a tight structure.ShSPI is the smallest Kazal functional domain that has been founded so far.The important residues that may affect the function of ShSPI were assayed by some point-mutation peptides.It was found that P2,Q3,P6,Y9,E25 are important residues for ShSPI to interact with elastase.In the experiment of plasma stability,ShSPI was able to maintain its high elastase inhibitory ability after incubation with plasma for more than 48 hours,which proves the high structural stability of ShSPI,In the formalin-induced mouse foot licking model,the group treated with ShSPI showed a significant analgesic effect whether in the neuropathic pain of phase I or the inflammatory pain of phase ?.Above all,ShSPI is an elastase inhibitor that offers a possibility for the development of analgesics.In the second part of this study,we obtained a 72-AA peptide named Joannsin(QAWQNYYRPSRAGYSYCYDDYDIGPCRARFRQWYYNRRTGECEIFFYGGCLGND NKYESKEECEYICKKLLT).Joannsin contains 6 cysteine residues and the results of sequence comparison showed that it belongs to the Kunitz family.Recombinant Joannsin was successfully expressed using prokaryotic expression system,and it was separated and purified by affinity chromatography,chemical cleavage by formic acid,Sephadex G50,and reverse high pressure liquid chromatography.Finally,a certain amount of high-purity Joannsin protein was obtained for subsequent experiments.In vitro enzyme-inhibiting activity screening showed that Joannsin can prolong APTT/PT time and have strong inhibitory activity on trypsin and FXa,which are serine proteases in the coagulation pathway.Joannsin could inhibit the activity of FXa to cut prothrombin into thrombin.Joannsin could not affect the activity of other serine protease of coagulation pathway,including thrombin,FXIIa,plasmin,chymotrypsin and kallikrein.After calculation,the Ki values of Joannsin for trypsin and FXa were 182.7 ± 14.6 nM and 29.5 ± 4.7 nM,respectively.In carrageenaninduced mouse tail thrombus model,Joannsin can inhibit thrombus formation obviously at the concentration of 2 mg/kg and Joannsin could affect strongly compared with the equimolar Apixaban,which is a small molecule of FXa inhibitor.Above all,the results in vivo and in vitro show that Joannsin has a strong inhibitory effect on the coagulation pathway.Joannsin could be used as a potential molecule for the clinical treatment of thrombotic diseases.In this part of the study,the active peptide component in the defensive glands of millipede was first reported.To a certain extent,this study enriches the database of peptides in arthropods,and improves the evolution system of arthropods.This paper demonstrates that serine protease inhibitors are present in S.hainanum and P.joannsi.ShSPI and Joannsin belong to the Kazal and Kunitz-type families,respectively.ShSPI with a new type structure of atypical Kazal family,while Joannsin has the classic Kunitz family structure.Different structures provide them different functions.The research makes a preliminary exploration of ShSPI and Joannsin in vitro and in vivo,which proposes the possibility that they can be used as therapeutic targets in different areas of disease.This research enriched the peptide database of arthropods,and provided important clues for the development of new clinical drugs which based on the active peptides.