Autoproteolysis And Substrate Analysis Of Subtilisin-like Protease 1 Of Nosema Bombycis

Microsporidia,a group of obligate intracellular unicellular eukaryotic parasites,have a wide host range and could infect almost all animals from invertebrate to vertebrate.Pebrine,Colony Collapse Disorder in honeybees and Slow Growth Syndrome in shrimp caused by microsporidia bring great economic loss to the breeding industry worldwide.The life cycle of microsporidia generally consists of infective phase(or environmental stage),proliferative phase and sporogonic phase.In infective phase,the only extracellular stage,mature spores are activated by environment factors,finally resulting in the spore germination with eversion and ejection of the polar tube,from where transports the infectious sporoplasm into host cell to achieve the invasion.The spore polar tube ejection of microsporidia is unique in nature,however molecular mechanism has not been clarified,which is also a significant scientific problem in the field of microsporidia.Subtilisin-like protease(SLP),a common virulence factor in pathogen,takes part in several biological events such as the formation of fungal appressorium,pathogen's release from host by processing substrates.Previous studies in our lab showed that subtilisin-like protease 1 of Nosema bombycis(NbSLPl)was located at anchoring disc.In addition,anchoring disc-spore wall complex protein NbSWP16 can interact with NbSLP1.We speculated that NbSLPl may contribute to spore germination by processing its substrate NbSWP16 in anchoring disc.In order to verify this hypothsis,here we focused on NbSLPl autoproteolysis,enzyme activity and its substrate.Main results are as followed:1.Identification of autoproteolysis and cleavage sites of NbSLPlThere exsits autoproteolysis of subtilisin-like protease containing inhibitor domain and catalytic domain,which means that the protease can produce mature enzyme through cleaving inhibitor domain.It is necessary to characterize autoproteolysis traits of NbSLPl in E.coli expression system.First,the sequence of Nbslp1 without signal peptide was cloned,and expression vector pET30-pro-Nbslp1 were constructed.After that,the vector was transformed into E.coli Transsetta(DE3).We purified the target protein from induced E.coli Transsetta[pET30-pro-Nbslp1]via Pull-down and SDS-PAGE,the results showed that the captured protein bands at size of?50 kDa and?15 kDa were consistent with the size of NbSLPl catalytic domain and Inhibitor_I9 domain.The LC/MS-MS analysis showed that both two protein bands were identified as NbSLPl,which demonstrated the autoproteolysis of NbSLPl precursor in E.coli.Furthermore,the N-terminal sequence of?50 kDa polypepitide were sequenced as NH2-Asp-Phe-Asn-Leu-Ser-Asp-Tyr-Arg-Ly s-Ly s,then P4-P4' self-cleavage sites of NbSLPl were deduced as Met101-Arg102-1le103-Ala104-Asp105-Phe106-Asn107-Leu108.The length of mature NbSLPl and NbSLPl-Inhibitor_I9 domain was 388 aa with MW 42.8 kDa and 77 aa with MW 8.8 kDa respectively.Based on this result,the self-cleavage site I103A104D105 was site-directed mutated to A103A104A105 by overlap PCR and the recombinant expression vector pET30-Nbslp1mut was constructed.Expression results showed that autoproteolysis of His-NbSLP1mut-His was reduced.2.Preparation of active enzyme of NbSLP1We designed the primers of Nbslpl-Peptidase_S8(Nbslp1S8)gene and constructed the expression vector pET30-Nbslp1S8.After that the plasmid was transferred into E.coli Transsetta.In the condition of 16? 80 r/min,the protein His-NbSLP1S8-His was induced by 0.3 M IPTG and expressed in the form of inclusion body,then the target protein was purified via affinity chromatography.After that on-column refolding was performed to obtain soluble protein.Folin-phenol method revealed that refolding protein showed very low enzyme activity.In order to obtain the mature enzyme of NbSLPl with high activity,E.coli Transsetta[pET30-pro-Nbslp1]were cultured in the condition of 16? 80 r/min for 20 h by incubating with 0.2 mM IPTG.After Purified by affinity chromatography,two his-tagged proteins,NbSLP1C-His and His-NbSLP119 were obtained.The detection result of enzyme activity of NbSLPl after desalting demonstrated that enzyme activity unit of NbSLP1C-His was 236.86 U/g,and His-Inhibitor_I9-domain could inhibit the enzyme activity of NbSLP1C-His in vitro.3.Substrate analysis of NbSLP1Based on NbSLP1 prokaryotic expression,we obtained the active mature enzyme of NbSLP1.Western blotting analysis of incubation of active NbSLPl and NbSWP16 in vitro showed that there was no different bands between control and experiment group,no evidence of NbSLP1 hydrolyzing NbSWP16 was obtained.Further,we prepared the co-transforming expression E.coli Transsetta[pET30-pro-Nbslp1]-[pET-DsbA-Nbswp16]to identify the proteolysis between NbSLP1 and NbSWP16 in bacterial cell,Western blotting analysis did not demonstrate that NbSWP16 was the substrate of NbSLP1.Hence,we have to explore the potential substrate of NbSLP1.According to the autoproteolysis motif MRIA104 ? D105FNL of NbSLP1 and sequence alignments of SLP1 homologues in microsporidia,results indicated that P4-P3' sites were conservative in Encephalitozoon,while P4,P2 and P1 sites were conservative in Nosema.Based on the conservative motif,screening of the potential substrates of NbSLPl in Nosema bombycis were conducted and some new candidates were explored.In general,this work identified the self-cleavaged sites of NbSLPl autoproteolysis and obtain the active mature enzyme of NbSLPl although we did not prove the enzyme and substrate relationship between the NbSLPl and NbSWP16.Now the new substrate candidates were screened according to the self-cleavaged sites of NbSLPl,we will carry out more experiments to elucidate the NbSLPl role in the polar tube ejection in Microsporidia in further work.