PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity

Abstract

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 μM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.