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

Abstract

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.