Cloning, Expression And Biological Function Of B Cell Stimulating Factor (fBAFF) And γ-IFN-induced Lysosomal Thiol Reductase (fGILT)

B-cell-activating factor of the TNF family (BAFF) induces B cell survival, proliferation, immunoglobulin secretion and plays a role in enhancing immune responses. In the present study, a BAFF homologue has been identified in fugu Takifugu obscures, and its biological activities have been characterized. The fugu BAFF (fBAFF) cDNA is 789 bp in length and translates into a 262-aa protein. The fBAFF genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size. Amino acid sequence comparison indicated that fBAFF possessed the TNF signatures, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the fsBAFF monomer analyzed by "comparative protein modelling" revealed that it was very similar to its human counterpart. Real-time quantitative PCR (qPCR) analysis revealed that fBAFF was predominantly expressed in fugu lymphoid tissue spleen. The SUMO-fsBAFF and GFP/fsBAFF efficiently expressed in Escherichia coli Rosseta (DE3) were confirmed by SDS-PAGE and Western blotting analysis. After purification, the GFP/fsBAFF fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsBAFF could bind to its receptors. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-fsBAFF could promote the proliferation of fugu splenocytes or mouse splenic B cells together with/without anti-mouse IgM. These findings indicate that SUMO-fsBAFF plays an important role in proliferation of fugu B cells and has functional cross-reactivity among fugu and other mammalians. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in fish. In mammals, interferon-y-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC classⅡ-restricted antigen (Ag) by catalyzing disulfide bond reduction. In the present study a fugu cDNA (designated as fGILT) was isolated from the spleen of Takifugu obscurus. It encodes a deduced protein of 242 amino acids with a putative molecular weight of 26.7 kDa, which has typical features of GILT proteins including the signature sequence CQHGX2ECX2NX4C, CXXC motif and other five cysteines responsible for the formation of disulfide bonds in the C-terminus. Genomic analysis revealed that fGILT gene, spanning a 1836 nt fragment, contained seven exons interrupted by six introns and exhibited a similar exon-intron organization to human and mouse GILT genes. Phylogenetic analysis showed that fGILT positioned at the base of vertebrate GILT clade, suggesting that it had been derived from a common ancestor with other vertebrate GILT proteins. The result of real-time PCR showed that fGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in spleen and kidney after induction with LPS. This study shows that fGILT may be involved in the immune response to bacteria challenge in Takifugu obscurus. It also provides the basis for investigating on the role of GILT using Takifugu obscurus as an animal model for human diseases.