Mechanism Of Inhibition Of Octodonta Nipae Prophenoloxidase Cascade By A Venom Protein,Tbserpin6,from Tetrastichus Brontispae

For successful parasitism,parasitoid wasps employ sets of strategies to impair host immune response.Venom from parasitoids especially that lack of Poly DNA Virus(PDV)has been validated to function in regulating host immune response,such as melanization.Immune response is an energy consuming process and accompany with production of toxic intermediates,which may harm host itself.However,serine protease inhibitors(serpins)can negatively regulate serine protease cascade and melanization.So far,lots of serpins and serpin-like proteases have been identified from venoms of parasitoids,they may play key role in the inhibition of activation of prophenoloxidase and melanization.In the present study,a venom protein,Tbserpin6,from Tetrastichus brontispae show a high similarity to Onserpin6 from Octodonta nipae,thus we speculated it may also inhibit the activation of host prophenoloxidase and melanization.To validate the hypothesis that Tbserpin6 function in inhibiting the activation of O.nipae prophenoloxidase and melanization,we cloned Tbserpin6 from T.brontispae,Onserpin6 and OnPPAF1(prophenoloxidase-activating factor 1)from O.nipae,and expressed their recombinant proteins.The main results in the present study are outlined below.1.Tbserpin6,Onserpin6 and OnPPAFl encoded 323,416 and 388 amino acids,respectively.Sequence alignment showed that OnPPAF1 is a kind of trypsin-like serine protease with typical "clip-domain" at N-terminal and serine protease domain at C-terminal.It belongs to CLIPS group which processes amidase activity and may have function in translating prophenoloxidase to active phenoloxidase.Tbserpin6 and Onserpin6 belong to serpin superfamily,which are highly similar to each other at activation sites P1-P1',Arg and Ile,respectively.We inferred that Tbserpin6 and Onserpin6 may have function in regulating the activation of O.nipae phenoloxidase.2.RNAi results showed that knockdown of OnPPAF1 caused grievous decrease of phenoloxidase activity and significant inhibition of hemolymph melanization,which suggests that OnPPAF1 have key function in melanization of O.nipae.3.Western blot results indicated that both Tbserpin6 and Onserpin6 interacted with OnPPAF1 to generate a covalent complex.Amidase activity assay indicated that both Tbserpin6 and Onserpin6 decreased the amidase activity of OnPPAF1.Hemolymph PO activity and melanization analysis once again confirmed our result that Tbserpin6 and Onserpin6 inhibited the melanization of O.nipae.In conclusion,our results revealed that OnPPAF1 played an important role in phenoloxidase cascade of O.nipae,while Tbserpin6 and Onserpin6 inhibited the activation of prophenoloxidase by inhibiting the activity of OnPPAFl and induced the down-regulation of downstream melanization.This may be one of the mechanisms that the T.brontispae venom regulates O.nipae phenoloxidase cascade.