Functions And Action Mechanisms Of Serine Protease SP1、SP13 And SP105 In The Innate Immune Response In Ostrinia Furnacatis(Guen(?)e)

Unlike vertebrates,most insects have no acquired immune system.They mainly rely on innate immune system for defending against the invasion from the various pathogens and parasites.Innate immune response is composed of humoral immunity and cellular immunity.Humoral immune response mainly includes the production of antimiacrobial peptides and melanization reactions.Thereinto,phenoloxidase plays a pivotal role in melanizationand it exists as a zymogen form-prophenoloxidase.The activation of prophenoloxidase results from the sequential activation of a series of serine proteases activation.Serine proteases,especially those with a clip domain,play important roles in melanization.Serine proteases are a group of proteolytic enzymes with serine residue as active centre.They consist of the clip domain at the N-terminus and a catalytic domain at the C-terminus.Catalaytic domain has a catalytic triad consisting of His,Asp,and Ser residues.Upon the infection from pathogen or parasite,a series of serine proteases are sequentially activated.Finally,the inactive prophenoloxidase(PPO)is converted into active phenoloxidase(PO)to initiate the melanin synthesis.Such extracellular serine protease cascades are often regulated by members of the serine protease inhibitor(serpin)superfamiliy.Serpin forms covalent complex with its cognate protease,and therefore,inhibits the protease irreversibly.The Asian corn borer,Ostrinia furnacalis(Gunee),is an important insect pest in Asia including China.In order to explore the molecule mechanisms involved in the innate immune response in O.furnacalis,we firstly checked the immune activities upon the infection,and determined that the innate immune response in O.furnacalis was indeed induced by the invaded bacteria.Then we selected 3 serine protease(OfSP1,OfSP13,OfSP105)and 3 serpins(serpin-3,serpin-6,serpin-11)for further studies.We investigated the relationships among them and their functions and functioning mechanisms with molecular biology and biochemical methods.The results were obtained as follow:1.The obvious melanization reaction in O.furnacalis was observed after the infection of Beauveria bassiana.And the PO activity in the hemolymph was elevated significantly upon the infection.2.The invasion of bacteria or fungi into O.furnacalis larvae increased the antibacterial activity of the hemolymph significantly.The transcriptal level of 3 antimicrobial peptide genes(OfgLys6,OfgAtt and OfgIID)were up-regulated significantly after the challenge.3.The full-length cDNA sequences of 3 serine protease genes(SP1,SP13,SP105)were cloned successfully.The deduced amino acid sequences were analyzed with bioinformatics methods.They showed distinct expression patterns with SP1 unchanged and SP13 and SP105 up-regulated.4.The recombinant SP1,SP13 and SP105 proteins were produced with Bac-to-Bac baculovirus system.The predicted activation sites in SP1,SP13 and SP105 were changed to IEGR,a preferred cleavage site for commercially available bovine Factor Xa.The obtained proSPlXa,proSP13Xa and proSP105xa could be cleaved and activated by Factor Xa.Thus,we obtained active SP1,SP13 and SP105 in vitro.5.During melanization process,SP1 cleaved proSP13,and therefore,functioned as upstream protease of proSP13.Active SP13 directly cleaved prophenoloxidase in vivo and in vitro,and therefore,functioned as a prophenoloxidase-activating protease(PAP).Meanwhile,SP1 failed to cleave proSP 105.Factor Xa-activated SP105Xa could cleave prophenoloxidase directly,suggesting SP105 was also a prophenoloxidase-activating protease(PAP).6.Serpin-3 and serpin-6 could form stable complexes with SP13 and SP105,and further inhibited the activation of prohenoloxidase by SP13 and SP105.Besides,serpin-6 also could form complex SP1.However,serpin-11 formed complex with neither one.It suggested that serpin-3 and serpin-6,other than serpin-11,had regulation on the melanization in O.furnacalis.Overall,we cloned 3 serine protease genes(SP1,SP13 and SP105)in O.furnacalis,investigated their functioning mechanisms in the melanization response,and explored the regulation by 3 serpins(serpin-3,serpin-6 and serpin-11).Our work thus revealed a biochemical mechanism in the melanization in O.furnacalis associated with the challenge by B.bassiana.This is the first report about the prophenoloxidase-activating protease in O.furnacalis.These insights could provide valuable information for better understanding the innate immune response in O.furnacalis and other insects.It could provide academic basis for the biocontrol of O.furnacalis by targeting at its innate immune system.