| X-box binding protein1(Xbp1), a basic leucine zipper transcription factor that found inhuman B cells, is a member of CREB/ATF proteins family. Xbp1is a key regulator ofunfolded protein response (UPR) signaling pathway. Xbp1is a dual coding gene whichcontains two overlapping open reading frames (ORFs), A and B. Under normal physiologicalconditions, only frame A is translated into ‘unspliced’ Xbp1protein, called Xbp1u. Uponsensing unfolded protein in the endoplasmic reticulum (ER) lumen, the transmembranesensor inositol requiring protein1(IRE1) removes26nucleotide (intron) from ORF A, andcleavage reroutes translation from5’ portion of frame A to B. This transcript uses acombination of two reading frames to produce a ‘spliced’ Xbp1protein-Xbp1s, as an activetranscription. Xbp1s will enter into nuclear, and up-regulate the expression of genesresponsible for protein folding, transporting, glycosylation and ER associated degradation.It had been confirmed that Xbp1also participates in innate immune reaction. Recentresearch has shown that Xbp1mRNA precursor has been transcriptional inducted aftertoll-like receptor (TLR) stimulation. Moreover, TLR signaling specifically activated theIRE1α-Xbp1branch in mouse macrophages without inducing canonical UPR. The activeXbp1s was shown to regulate the expression and secretion of key cytokines, such asinterleukin-6(IL-6), interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α). Theenvironment fish living in is conducive to bacteria and viruses survival. Any damage of fishwill cause pathogens to drive straight through the wounds of skin or gut into the body andthreaten fish. So far, limited studies have reported the functions of Xbp1in teleost. Xbp1scontributes to identify terminally differentiating B cell populations and regulate UPR. Thereis no any research about carp Xbp1or the innate immune role of Xbp1in fish.Here we identified two types of carp Xbp1, Xbp1u and Xbp1s. Carp Xbp1contains twooverlapping ORFs. ORF A contains792bp and encodes263amino acids. ORF B contains609bp and encodes202amino acids. Carp Xbp1u and Xbp1s have identical168amino acids at N-terminal region but variable C-half region.Xbp1is a highly conserved protein distributing from yeast to higher mammals. Here wepredicted the secondary structure of carp Xbp1sequence around the IRE1cleavage sites usingCentroidFOLD. It formed two stem-loop structures. Multiple alignments of the sequencesabove showed that they were well-conserved in all species. The bZIP region is the onlycomplex domain in carp Xbp1protein. There is a significant homology of amino acidsequence between the carp Xbp1protein and other species. There was a conserved bZIPregion in Xbp1. The phylogenetic tree showed that carp Xbp1is a closer match to zebrafishXbp1. Above all, we concluded that unconventional splicing of the bZIP-containing protein isevolutionarily old, present in the last common ancestor of Metazoa and Fungi and understrong selection in the majority of species.Real-time PCR results showed that two carp Xbp1transcripts were ubiquitouslyexpressed in all tissues. The expression of Xbp1u was higher than the spliced one underphysiological state.To determine the role of carp Xbp1in innate immunity, we stimulated carp withpathogenic bacteria V.anguillarum, and then analysed the expression of Xbp1mRNA indifferent immunologic tissues by Real-time PCR. The results showed that the functionalXbp1s mRNA levels both increased gradually and significantly in skin, gill, foregut, hindgut,headkidney, spleen, and liver from0hour to48hour after challenge with pathogenic bacteriaby either soak or injection. In contrast, expression level of total Xbp1mRNA did not change.We also examined tlr2, tlr4, and ifnγ mRNA levels in headkidney and hindgut after infectionat0hour to12hour. All of them were increased as we expected. Our study implied that carpXbp1plays a role as other species Xbp1in innate immune. The mechanism of interactionbetween carp Xbp1and TLR still need further research. The studies about carp Xbp1will notonly enrich the fish Xbp1data, but also provide a theoretical basis for prevention andtreatment of fish diseases. |
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