Study On Expression And Function Of A Pattern Recognition Serine Protease (EsPRSP) And A Serine Protease Inhibitor (Esserpin) From Crab Eriocheir Sinensis

Chinese mitten crab Eriocheir sinensis is one idiomatical crustacean species inChina, possessing crucial economic and scientific value. As the unique immunecomponent of invertebrate innate immunity, the prophenoloxidase-activating systemplays key roles in the immune defense response of Chinese mitten crab. In the presentstudy, the expression profile and function of two genes from E. sinensis (patternrecognition serine protease designated as EsPRSP, and serine protease inhibitordesignated as Esserpin) were investigated by the techniques of molecular biology, cellbiology and bioinformatics based on their full-length cDNA sequences.The mRNA transcripts of EsPRSP could be detected in all the examined tissuesincluding heart, gill, hemocytes, muscle, cuticle and hepatopancreas, and the highestexpression level was present in hemocytes which was7.17-fold (P <0.05) of that inheart. While the expression of EsPRSP in hepatopancreas, muscle, gill were3.18,2.74,and1.63-fold of that in heart, respectively. But there was no significant difference (P>0.05) between cuticle and heart. After the crabs were challenged by Vibrioanguillarum and Pichia pastoris, the expression levels of EsPRSP transcripts inhemocytes were significantly up-regulated, and peaked at24h (2.31-fold of blankgroup, P <0.05) and12h (2.56-fold of blank group, P <0.05), respectively. Therecombinant EsPRSP (rEsPRSP) could inhibit the growth of E. coli and V.anguillarum to some extent at exponential phase, showing antibacterial activity. In thepresence of rEsPRSP and V. anguillarum, PO activity in the plasma was significantly up-regulated compared with the blank plasma. V. anguillarum could induce theproduction of associated factors of prophenoloxidase-activating system and they werenecessary to EsPRSP functionating.The mRNA transcripts of Esserpin could be detected in all the examined tissuesincluding heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highestexpression level was present in gonad (62.50-fold, P <0.05) and muscle (9.93-fold, P<0.05), compared with that in hemocytes. But there was no significant difference (P>0.05) in heart, gill and hepatopancreas, compared with that in hemocytes. After thecrabs were challenged by V. anguillarum and P. pastoris, the expression levels ofEsserpin transcripts in hemocytes were significantly up-regulated, and reached thepeak level at24h (5.18-fold of blank group, P <0.05) and3h (2.87-fold of blankgroup, P <0.05), respectively. The recombinant Esserpin (rEsserpin) could inhibittrypsin activities in a dose-dependent manner, and it could lead to100%inhibition oftrypsin activities under the concentration of873.76nM, while there was no evidentinhibition of chymotrypsin observed with rEsserpin. The rEsserpin could also inhibitthe growth of E. coli and V. anguillarum to some extent. In the presence of rEsserpin,PO activity in the plasma was significant (P <0.05) lower than that of the blankplasma and the rTrx treated plasma. These results indicated that Esserpin was ahomologue of serpin in crab and it could be induced after immune stimulation andmediate immune response possibly via the inhibition of bacterial growth and theregulation of prophenoloxidase-activating system.All the results demonstrated that pattern recognition serine protease (EsPRSP) andserine protease inhibitor (Esserpin) were involved in the immune defense response ofChinese mitten crab and played important roles. This study provided new insights intothe molecular basis and regulation machnism of invertebrate proPO system, as well asdeveloped the knowledge of invertebrate’s immunology.