Molecular structure, expression, and bioactivity of B-cell–activating factor of the TNF family (BAFF) and its receptor BAFF-R in cats (Felis catus)

• Published in: Molecular immunology Vol. 112; pp. 59 - 71
• Main Authors: Sang, Ming, Li, Jianfeng, Wei, Zhiheng, Wu, Xiaolong, Wang, Zhiguo, Ma, Lei, Liu, Hongzhen, Zhang, Shuangquan, Zhang, Jiaxin
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2019
• Subjects: BAFF; BAFF-R; Cat; Immunology function; Ligand–receptor interactions; Molecular structure; BAFF; Molecular structure; IgG; csBAFF; EGFP; TNF; IgM; BAFF-R; SDS-PAGE; SAC; RT-PCR; Cat; IPTG; PI; cDNA; GAPDH; Immunology function; Ligand–receptor interactions
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2019.04.031

•Cat (Felis catus) BAFF and its receptor BAFF-R were cloned.•csBAFF, EGFP/csBAFF, and cBAFF-R were expressed in E. coli and purified using a Ni2+ column.•csBAFF cellular localization was assessed.•csBAFF promoted B-cell survival in vitro.•Ligand–receptor interaction was assessed. B-cell survival depends on signals induced by binding of B-cell activating factor (BAFF) to its receptor (BAFF-R). In this study, the full-length cDNAs of cat BAFF (cBAFF) and BAFF-R (cBAFF-R) were amplified from the spleen by reverse transcription PCR. The open reading frame of cBAFF cDNA encodes a protein of 285 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFFs. The cBAFF-R gene encodes a 189 amino acid protein. Real-time quantitative PCR analyses revealed that the two genes are predominantly expressed in the spleen. csBAFF, EGFP/csBAFF, and cBAFF-R were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. After purification, the EGFP/csBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Confocal laser scanning microscopy showed that EGFP/csBAFF bound to its receptor. In vitro, csBAFF promoted the survival of cat and mouse splenic B cells with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, it stimulated the survival of mouse B cells, similar to msBAFF. Recombinant cBAFF-R blocked the function of sBAFF in vitro. These findings indicate that csBAFF plays an important role in the survival of cat B cells and has functional cross reactivity between cats and other mammals, and suggest a role for the BAFF–BAFF-R system in regulating B-cell survival. Therefore, BAFF and BAFF-R show promise for enhancing the immune systems of animals.