Cloning And Characterization Of The Initiator Caspase-5 Of Spodoptera Litura

Apoptosis is a normal programmed cell death,Caspase family play an important role in the mitochondrial apoptosis pathway in mammals.Caspase family can be divided into two groups,initiator caspase and effector caspase.Generally,the activated initiator caspase can cleave and activate effector caspase,resulting in the caspase cascade.Therefore,the initiator caspase play a very important role in apoptosis pathway.Several initiator caspases have been identified in mammals.Caspase-9 among them is an initiator caspase of more in-depth study.To date,the homologous genes of caspase-9 in the Lepidoptera insects such as Bombyx mori,Gypsy armyworm and Spodoptera frugiperda,have also been identified.The studies of apoptosis in Spodoptera litura have been carried out in our laboratory for many years.In order to deeply investigate a apoptosis signal transduction pathway in S.litura,we have cloned a Sl-caspase-5 gene,which is the caspase-9 homologue from S.litura,expressed Sl-caspase-5 protein in prokaryotic cells,prepared the antibody against Sl-caspase-5 proteins and preliminarily studied the function of Sl-caspase-5.The main research results are as follows:1.Cloning of Sl-caspase-5 gene,the analysis of the amino acid sequence alignment and the prediction of protein function(1)The Sl-caspase-5 gene successfully cloned is 1587bp in length and the largest open reading frame is 1338bp and encodes a predicted protein of 445 amino acids with isoelectric point of 6.40 and a molecular mass of 51.136Da.(2)The alignment of predicted amino acids of sequence of Sl-caspase-5 with initiator caspase orthologs of B.mori,Helicoverpa armigera,Spodoptera frugiperda,Lymantria dispar,and Drosophila melanogaster showed that Sl-caspase-5 shares 54%,70%,88%,58%and 28%amino acid identity with BmDranc,Ha-caspase-5,SfDranc,Ld-caspase-5 and DmDranc.There are 14 a helixes,5 ? folds and some random coils in predicted tertiary structure of Sl-caspase-5 protein.Sl-caspase-5 possess the characteristics of initiator caspases including QMCRG sequence,a conservative five peptide sequences around the cysteine(C)at 310 site,recruitment domain CARD and the activated domain consisting of P20 and P10 subunits.Structure analysis shows that Sl-caspase-5 belongs to initiator caspases.2.Prokaryotic expression and purification of Sl-caspase-5Recombinant plasmid pET-28a-Sl-caspase-5-His6 was constructed and transformed into BL-21 cells to express the Sl-caspase-5 proteins.The proteins expressed were purified by using Ni NTA column and molecular sieve purification system respectively.The Sl-caspase-5 protein expressed in prokaryotic expression system could self-cut to cause the self activation.In order to obtain the whole protein,the prokaryotic expression of the point mutation of Sl-caspase-5 was carried and the full proteins were obtained and used to make the polyclonal antibody.3.Sl-caspase-5 has the initiator caspases enzyme activityWhen Sl-caspase-5 was expressed and purified,the Sl-caspase-5 protein could cleave itself and cause the activation.The activity of Sl-caspase-5 was detected by using an Ac-IETD-AFC fluorescence as substrate,which is mammalian caspase-8's substrate.Sl-caspase-5 could effectively cleave Ac-IETD-AFC,indicating that the Sl-caspase-5 has the enzyme activity of initiator caspase.4.The functional analysis of Sl-caspase-5 protein(1)The interaction among Sl-caspase-5,Apaf-1 and the effector Sl-caspase-1 were performed in vitro.The activated Sl-caspase-5 could cleave Sl-caspase-1 protein which was cut into two subunits with molecular mass of about 19 KD and 12KD by SDS-PAGE.The inactive Sl-caspase-5 zymogen protein was expressed under the condition of low temperature at a short time.In the presence of dATP and inactive Sl-caspase-5 zymogen,the truncation AF-627(activated Apaf-1)could activate Sl-caspase-5 zymogen protein.Then actived Sl-caspase-5 cleaved Sl-caspase-1 into two subunits.This result is consistent with above alone treatment with activated Sl-caspase-5.When Sl-caspase-5-Card protein added to the above system,the cleavage of Sl-caspase-1 was not observed,hinting that truncated Sl-caspase-5-Card protein interacts with Apaf-1 competitively,thus it likely inhibit Sl-caspase-5 being actived.So Apaf-1 is required for the activation of Sl-caspase-5.(2)Over expression of Sl-caspase-5 induced apoptosis in Sl-1 cellsApoptotic bodies were observed in the SI-1 cells with transfected pIE2-N1-Sl-caspase-5-GFP under fluorescence microscope after 36 hours.No apoptotic bodies were found when the over expression of Sl-caspase-5-C310A but the accululation phenomenon of green fluorescent protein of caspase-5-C310A was observed in cells.It was due to the Sl-caspase-5-C310A active site cysteine was mutated,so it can be recruited by Apaf-1 and but can not be activated and not cause the downstream cascade reaction.It suggests that over expression of Sl-caspase-5 can induce an apoptosis in Sl-1 cells.3)Over expression of Sl-caspase-5 promotes apoptosis induced by actinomycin DSl-1 cells were transfected with eukaryotic expression plasmids including pIE2-Sl-caspase-5-Flag-N1,1 pIE2-Sl-caspase-5-Card-Flag-N 1,pIE2-Sl-caspase-5-C31 OA-Fla g-N1,pIE2-Flag-N1 respectively after treated with a low concentration of actino mycin D.The cleavage of Sl-caspase-1 was detested by Western blotting only wh en over expression of Sl-caspase-5.The analysis of flow cytometry showed that apoptotic rate was 40.05%,20.09%respectively when transfected with pIE2-S1 caspase-5-Flag-N1 and empty plasmid and apoptosis rate was 13.48%and significantly lower than that of the other groups when the over expression of Sl-caspase-5-Card.It shows the over expression of Sl-caspase-5 can promote the apoptosis induced by actinomycin D in Sl-1 cells.The cloning,identification and functional analysis of S.litura initiator caspase Sl-caspase-5 lay the foundation for the relationship among Sl-caspase-5 cytochrome c and Apaf-1,formation and function of apoptotic body and cytochrome-mediated mitochondrial pathway.