Molecular Structure Characteristics And Function Analysis Of HsLITAF In Hyriopsis Schlegelii

Lipopolysaccharide induced TNF-a factor(LITAF)plays an important role in innate immune system of invertebrates.In this study,the cDNA full-length sequences of three HsLITAFs were obtained by RACE-PCR according to their partial EST sequence and named as HsLITAF1,HsLITAF2 and HsLITAF3.The full length of their cDNA were 720 bp,1115bp and 688 bp,respectively,encoding 146,163 and 115 amino acid proteins.The theoretical molecular weight were 16.17 for HsLITAF1,17.41 for HsLITAF2 and 12.48 for HsLITAF3.Analysis of the three genes revealed that there were 31% to 94% homology and 48% to 100% similarity compared with other species.Except HsLITAF3,the other two subtypes had two conserved CXXC motifs.Phylogenetic tree and protein structure analysis were conducted to determine their identities and evolutionary relationships,which showed that HsLITAFs members clustered into two different mollusk LITAF branches.The mRNA transcription levels of three HsLITAFs were detected in hemocytes,gill,hepatopancreas,mantle and kidney.Results showed that three HsLITAFs could be detected in all tissues,HsLITAF1 and HsLITAF3 were with the highest expression level in hepatopancreas,while Hs LITAF2 with the highest expression level in gill.Meanwhile,we observed all Hs LITAFs were with the lowest expression in hemocytes.However,we paid closely attention to the expression of three HsLITAFs after stimulation by LPS at 3 h,6 h,12 h,24 h,48 h in hemocytes and the data showed that the expression of all HsLITAFs were up-regulated at 3 h,HsLITAF1 and HsLITAF3 showed the highest expression at 24 h,HsLITAF2 showed the highest expression at48 h.It's implied that HsLITAFs were involved in the immune response of LPS challenged.CCK8 detected the proliferation of SMCC-7721 cells in vitro showed that HsLITAF1,HsLITAF2 and HsLITAF3 genes inhibited the proliferation of cells in vitro.The rate of cell proliferation after transfection of HsLITAFs were 71.46%,72.20%and 77.06%,respectively.Flow cytometry analysis showed that the apoptosis rat of SMCC-7721 were 46%,24.98% and 12.84%,respectively.In three tested group,the results showed that the expression of Bax was up-regulated 1.43,1.41 and 1.29 fold,and the Bcl2 was down-regulated to 0.33,0.32 and 0.59 fold respectively.TheCaspase3 was up-regulated 1.80,3.05 and 3.05 fold respectively,P53 was up-regulated 7.04,7.92 and 3.10 fold,respectively.It's implied that three HsLITAFs might induce the apoptosis of SMCC-7721 cell by regulated the expression of those apoptosis-relative gene.The bioinformatics prediction of subcellular localization of three HsLITAFs found that HsLITAF1 was most likely to be located in the nucleus with a possibility69.6%.HsLITAF2 was most likely to be located in the nucleus with a possibility38.4%.HsLITAF3 most likely located in the nucleus with a possibility 69.6%.However,the subcellular localization results showed that HsLITAF1 and HsLITAF2 were expressed in the nucleus and cytoplasm,and were mainly expressed in the nucleus.While,Hs LITAF3 was expressed only in the cytoplasm,which indicated that the prediction of bioinformatics was not exactly accurate.HsLITAF3 could not enter the nucleus might related to its loss of C-terminal CXXC structure,and lead HsLITAF3 and HsLITAF1 with a functional differences.All in all,the results showed that the three HsLITAFs were involved in the innate immune response and showed different responses after LPS stimulation.All HsLITAFs could induce the apoptosis of SMCC-7721 cells through regulated the expression of apoptosis-relative gene.HsLITAF3 can not be nucleated in the absence of CXXC domain,which might lead to a decrease in its cytotoxicity.