Preliminary Study On Identification And Function Of SPSB Family In Oyster Crassostrea Gigas

The SPSB(SPRY domain containing SOCS box)family refers to a cluster of molecules containing a typical SPRY domain and a typical SOCS box domain.In vertebrates,the SPSB family is consisted of four members,SPSB1-SPSB4,which could mediate NO synthesis,regulate the production of interferon by interferon regulatory factors,and plays important roles in life activities such as growth and development and immune defense.In invertebrates,the SPSB family has also been identified from several species such as drosophila.However,the biological functions of SPSB family,especially its roles in innate immune regulation,are still poorly studied.In the present study,the molecular composition,interactions with inducible nitric oxide synthase(iNOS),and synthesis induction of cytokines including interleukin-17(CgIL17-5),tumor necrosis factor(CgTNF-1)and interferon-like protein(CgIFNLP)of SPSB was studied in the Pacific oyster Crassostrea gigas,hoping to illustrate the molecular components and involvement in innate immunomodulation of SPSB family in oyster using the methods of molecular biology,cell biology,bioinformatics,and so on.The SPSB family in C.gigas consisted of two members,CgSPSB1 and CgSPSB3.The open reading frames of CgSPSB1 and CgSPSB3 genes were 837 bp and 699 bp,respectively,encoding polypeptides of 279 and 232 amino acids.The domains of CgSPSB1 and CgSPSB3 were highly conserved and each contained a typical SPRY domain and a typical SOCS box.CgSPSB1 shared more than 70% sequence similarity to model organisms such as human,mouse,zebrafish,and drosphila.In the phylogenetic tree,SPSB1 and SPSB4 clustered into a cluster in higher animals,and then clustered with CgSPSB1 of C.gigas,and finally gathered with GUSTAVUS(GUS)of Drosophila.CgSPSB3 shared more than 50% similarity with SPSB3 sequences in invertebrates such as C.virginica,Mizuhopecten yessoensis,Limulus polyphemus and Drosophila obscura.But shared low similarity with SPSB3 in vertebrates.In the phylogenetic tree,CgSPSB3 was clustered with SPSB3 in other species,and SPSB1/2/4 is a large branch.The mRNA transcripts of Cg SPSB1 and CgSPSB3 could be detected in all the tested tissues of C.gigas,and the highest expression levels were observed in hemocytes.Results of the immunofluorescence analysis showed that the CgSPSB3 protein was mainly expressed in the cytoplasm of oyster hemocytes.At 12 h after lipopolysaccharide(LPS)stimulation,the mRNA expression of CgSPSB1 and CgSPSB3 in hemocytes was significantly up-regulated,which was 2.06-fold and 2.71-fold of that in the control group(p < 0.05).Besides,the mRNA expression of CgSPSB3 in hemocytes was up-regulated at 3 h after Vibrio splendidus stimulation,and peaked at 24 h,which was about 5.03-fold(p < 0.05)of that in the control group.In order to investigate the interaction of recombinant CgSPSB1 with recombinant CgNOS(1-370aa)using pull down method.It was found that rCgSPSB1 could not bind to rCgNOS(1-370aa)in vitro.Further analysis of bioinformatics revealed that the SPRY domain of CgSPSB1 contained a conserved sequence Tyr133 which recognized the D-I-N-N-N sequence in iNOS,whereas CgSPSB3 does not.And the N-terminus of CgNOS did not contain the D-I-N-N-N amino acid sequence which was indispensable for CgSPSB1 binding.These results suggested that SPSBs could not induce NO production in oyster.In the present study,it was found that both rCgSPSB1 and rCgSPSB3 could interact with oyster interferon regulatory factor(CgIRF-8),and knock-down of CgSPSB3 with RNAi could result in decrease of mRNA expression of CgIFNLP,CgIL17-5 and CgTNF-1.These results indicated that SPSBs in oyster,similar to their homologues in vertebrates,were also capable of prompting the biosynthesis of cytokines such as IL,TNF and IFN.In summary,the CgSPSB1 and CgSPSB3 in C.gigas were conserved,which contained a typical SPRY domain and a typical SOCS box.Both rCgSPSB1 and r CgSPSB3 could interact with CgIRF-8,and CgSPSB3 could regulate the production of cytokines such as IFN,IL17 and TNF in hemocytes.CgSPSB1 could not regulate the production of NO by interacting with CgNOS,which owing to the lack of D-I-N-N-N sequence in CgNOS.This study preliminarily elucidated the role of the oyster SPSB molecule in the innate immune response,and also provided a reference for the immunoregulatory mechanism of the SPSB family of invertebrates.