Characterization, Expression And Functional Analysis Of Trypsin Gene In Amphioxus Branchiostoma Japonicum

Trypsin is a kind of main alkaline proteolytic enzymes in alimentary canal. It belongs to the family of serine proteinase, and specially hydrolyzes the peptide bond formed by Lysine or Arginine. Trypsin plays a central role in pancreatic exocrine physiology because it acts as the trigger enzyme for the activation of all other pancreatic digestive zymogens, as well as its own in active precursor, trypsinogen. Trypsin is widely distributed in organisms ranging from invertebrates to vertebrates. However little is known to date about trypsin in the amphioxus Branchiostoma japonicum. Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. In this study, we report the identification expression and functional analysis of trypsin gene in amphioxus B. japonicum.The cDNA encoding B. japonicum trypsin, Bjtry, was obtained by RACE-PCR. It is 978 bp long. Its longest ORF encodes a 272 amino acids long protein with a predicted molecular mass of approximately 29 kDa, with a 15 amino acids long N-terminal signal peptide and a 16 amino acids long activation peptide. The BLASTp searching at NCBI showed that the deduced protein had a Tryp_SPc domain at the residual positions 32-259 and a catalytic triad His72-Asp118-Ser215 which are involved in the catalytic mechanism of serine proteinase. It also had the highly conserved residue Asp209 at the bottom of the substrate binding pocket necessary for proper folding of the catalytic site and Tyr190 involved in the enzyme substrate specificity as well as six conserved cysteine residues (57-73, 184-200, 211-235) forming three disulfide bonds. The phylogenetic tree constructed using trypsinogens showed that amphioxus trypsinogens and ascidians trypsinogens grouped together, forming an independent cluster, which branched off from vertebrate trypsinogens.For investigating the function of BjTRY protein, the recombinant plasmid has been constructed, transferred to E.coli and induced to express. BjTRY expressed in insoluble body with a abundant amount. We denatured the insoluble body and purified it. Using the trypsin specific substrate BAEE we detected the enzyme activity of recombinant BjTRY, and got an enzyme activity of 375 BAEE U/mg. After that,the effect of some protein inhibitors on the recombinant BjTRY to BAEE was studied, and it shows that BjTRY enzyme activity can be inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor and the serine proteinase inhibitor PMSF, respectively, which further indicates the recombinant BjTRY is a kind of trypsin belonged to the serine proteinase super family.Northern blot, In situ hybridization demonstrated that Bjtry transcript was abundant hepatic caecum, the mid-gut and hind-gut, and at a slightly lower level in the ovary. Whole mount in situ hybridization showed that Bjtry transcript was first appeared in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Comparison to the expression of vertebrate trypsin gene and related genes in the pancreas suggests that the vertebrate pancreas is homologous to an extensive region of the amphioxus middle digestive tract that contains the hepatic caecum and extends posterior to mid-gut. This proposed homology implies that the vertebrate pancreas probably evolved by an extensive pre-existing middle region of digestive system including the hepatic caecum and mid-gut of an amphioxus-like ancestor.