Characterization and functional analysis of interleukin-6 and its receptor subunits (IL-6Rα and IL-6Rβ) in the yellow drum, Nibea alibiflora.

Abstract

Interleukin 6 (IL-6) is one of the cytokines found to be multifunctional and biologically effective, regulating immune and inflammatory response by interacting with receptors to transmit signals. In this study, the full-length cDNAs of IL-6 (named as NaIL-6) and its receptors IL-6R and gp130 (named as NaIL-6Rα and NaIL-6Rβ) of Nibea albiflora were acquired and they possessed the typical symbolic motifs similar to its teleost orthologues in multiple sequence comparisons. The phylogenetic trees showed that NaIL-6 and its receptors clustered with their counterparts in bony fish, and had the closest affinity to Larimichthys crocea. Real-time PCR indicated that NaIL-6, NaIL-6Rα and NaIL-6Rβ were widely expressed in different tissues, among which NaIL-6 was highly expressed in the liver, NaIL-6Rα showed the highest expression in the kidney and NaIL-6Rβ was reflected in the liver. Following stimulation by Vibrio parahaemolyticus, Vibrio alginolyticus, or Polyinosinic-polycytidylic acid (Poly (I:C)) infection, the mRNA expression of all three genes were greatly up-regulated over time. The cell localization analysis showed that NaIL-6 distributed in cytoplasm and cell membrane, and NaIL-6Rα located on the cell membrane. After co-transfection of NaIL-6- mCherry and NaIL-6Rα-EGFP, they co-expressed as orange at the same position for their possibility of spatial interactions on the cellular membrane. By GST-Pull down with the purified target proteins, the association between NaIL-6-His and NaIL-6Rα-GST was confirmed, which may be utilized for further functional study. Taken together, the results indicated that the biological functions of NaIL-6 and its receptors NaIL-6Rα and NaIL-6Rβ involved in the immunologic mechanism in N.albiflora, which would provide a basis for the research of immune functions and signaling pathway of IL-6 and its receptors in fish.