Study On The Immune Response Patterns,Enzymatic Properties And Apoptosis Inducing Functions Of Three Cathepsins From Scylla Paramamosain

Scylla paramamosain is an important farming breed found along the southeastern coast of China with high nourishment value and economic value.However,problems with microorganism infection have heavily impacted on commercial S.paramamosain farming.In recent years,the importance of disease control has attracted numerous attention about the immune mechanism of S.paramamosain.Cathepsins as members of lysosomal proteases are involved in many physiological activities such as intracellular protein degradation,zymogen or prohormone activation,immune regulation,inflammatory response,and extracellular matrix remodeling.Thus studying the functional properties of cathepsins of S.paramamosain will contribute to understanding the innate immune systems of crustaceans.So far the studies about cathepsins mainly concentrate on mammals while studies on crustaceans are very limited.In our previous study,three partial cDNA sequences exhibited certain sequence homology with cathepsin B,L and D were obtained from the embryo transcriptome library of S.paramamosain,and were named as SpCathB,SpCathL and SpCathD.The main findings are shown as followed:1.The open reading frame(ORF)sequences of SpCathB,SpCathL and SpCathD were obtained and their amino acid sequences were analyzed by bioinformatic methods.All three proteins were composed of a signal peptide,a propeptide domain and a mature domain.999 bp ORF sequence of SpCathB encoded 322 amino acids,and its amino acid sequence contained 3 cysteine protease active cites.1029 bp ORF sequence of SpCathL encoded 342 amino acids,and its amino acid sequence contained 3 cysteine protease active cites.1158 bp ORF sequence of SpCathD encoded 385 amino acids,and its amino acid sequence contained 2 aspartic protease active cites.2.The distribution of SpCathB,SpCathL and SpCathD in normal tissues and the expression pattern of SpCathB,SpCathL and SpCathD induced by Vibrio Parahaemolyticus infection and LPS challenge in vivo were elucidated.The three genes showed similar expression characterisitics.They were detected in all collected tissues of both male and female S.paramamosain while the highest expression levels of them were present in the hepatopancreas and the mid-gut.As for the V.Parahaemolyticus infected and LPS challenged group,the expression levels of SpCathB,SpCathL and SpCathD were found to be decreased in hepatopancreas,mid-gut and hemocytes.These results indicated that cathepsins may be involved in the immune response of S.paramamosain.3.The purified proSpCathL protein was obtained by eukaryotic expression and its enzymatic properties were determined.The recombinant proSpCathL protein was successfully expressed in Pichia pastoris and purified by Ni-NTA column.Autocatalysis of proSpCathL into a mature peptide was found to occur in acidic condition.The enzyme activity assay also showed that SpCathL exhibited cathepsin L activity and its optimum pH is 5 while its optimum temperature is 30?.4.The apoptosis related functions of SpCathB,SpCathL and SpCathD were explored.The co-locolization assay showed that SpCathB,SpCathL and SpCathD could enter into lysosomes after overexpression in insect cells.TUNEL detection showed that SpCathB,SpCathL and SpCathD could not directly induce apoptosis of insect cells but could aggravate apoptosis by co-transfection with Sp-caspase.Moreover,SpCathL was found capable of degradating Sp-caspase but could not acitivate the latter,which indicated that cathepsins may regulate apoptosis through other ways other than directly activate caspases in S.paramamosain.In conclusion,on the one hand the basis characteristics of SpCathB,SpCathL and SpCathD were indentified in this study,including gene cloning,bioinformatic analysis,tissue distribution analysis,intracellular localization detection,eukaryotic expression and enzymatic property analysis.On the other hand,the immune responses and apoptosis related functions of SpCathB,SpCathL and SpCathD were explored,which proved that cathepsins are important immune factors in S.paramamosain.This study would provide constructive guidance for further research of cathepsins and invertebrate immune system.