Functional Study Of Protease Inhibitor And Protease Of Thelohanellus Kitauei

Myxospores,the main parasitic protozoan of fish,cause serious damage to the production of fisheries.At present,the prevention and treatment measures of myxosporidiosis mainly rely on chemical reagents,which not only cause residual drug residues in fish,but also cause water pollution.Therefore,it is particularly important to clarify how the parasite invades and infects the host fish from molecular mechanisms.The previous study of the Genomics and transcriptome highlights the importance of secreted protease inhibitors and secreted proteases in nutrient metabolism and tissue invasion.Based on the transcriptome data of the early Myxospores Thelohanellus kitauei,the secreted cysteine protease inhibitor TK Cystatin was successfully cloned from 19 secreted protease inhibitor genes.TK Cystatin encodes a cysteine protease inhibitor with a gene length of 363 bp encoding 120amino acids and a theoretical isoelectric point PI of 6.97.ClusterW multiple sequence alignment revealed that TK cystatin has a conserved site for cysteine protease inhibitors,with N-terminal G¹⁹G²⁰,Q⁷³VVAG⁷⁷,and C-terminal L¹⁰²P¹⁰³ motifs.Through the construction of phylogenetic tree,the protein is closely related to the cystatin-A protease inhibitor of Hydra vulgaris,supporting for the Myxospore belong to the Cnidarian.The activity of TK Cystatin was detected by fluorescent peptide substrate assay.TK Cystatin inhibited the standard proteases papain,cathepsinL and cathepsinB.The IC₅₀ of TK Cystatin to papain,cathepsinL and cathepsinB were 0.8μmol/L,0.175μmol/L and 2.1μmol/L respectively,the inhibition constant Ki for papain was 0.88μmol/L.The inhibitory activity of TK Cystatin did not change much at 20℃-47℃,and showed a certain temperature stability.It was found that TK Cystatin has strong inhibitory activity under alkaline conditions.The Pulldown assay shows the interaction of TK Cystatin protein with Crap precathepsinL in the intestinal tract of carp.Remove the signal peptide from the gene then heterologous expression and purification yielded CC cathepsinL,but protease activity of CC cathepsinL is so low.Further,three secreted protease genes,TK Papain-1,TK Papain-2 and TK Pepsin,are successfully cloned and expressed from 34 secreted protease genes.TK Papain-1 and TK Papain-2 are found by bioinformatics analysis belong to the papain（C1A）family and encodes cysteine proteases encoding 150and 322 amino acids,respectively.The protein sizes are 17.06 kDa and 36.7 kDa,and the theoretical isoelectric points are 9.30 and 7.75,respectively.TK Pepsin belongs to aspartic protease,encoding 491amino acids,the theoretical size of the encoded protein is 55.41 kDa,and the theoretical isoelectric point is 6.37.All four proteins have signal peptide sequences,which are secreted proteins;only TK Papain-1 has Disulfide bond.Through the construction of the phylogenetic tree,it is found that the genetic relationship is close with the nematostella vectensis,Hydra vulgaris and ciliates.Also provides support data for Myxospore to the Cnidarian.Since these proteins are insoluble,this limits the study of their properties and functions.This study provides a basis for further research on the function of protease inhibitors and proteases of Myxospore and the interaction mechanism between Myxospore and host.