Molecular characterization and immune responses of Rab5 in large yellow croaker (Larimichthys crocea)

Rab5 regulates key steps in membrane traffic transport and endocytic pathway of host immune responses. In the present study, a full-length cDNA of Rab5 (LcRab5) was cloned from large yellow croaker, Larimichthys crocea and contained an open reading frame (ORF) of 648 bp encoding a 215 amino-acid protein. The amino acid sequence identities between LcRab5 and the homologues in mammals and other fish are from 87% to 97%. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcRab5 mRNA was predominantly expressed in blood and gill. The expression levels of LcRab5 were investigated in liver, spleen, and head-kidney after fish were challenged with LPS, polyinosinic polycytidynic acid and Vibrio parahaemolyticus. LcRab5 transcripts were significantly induced in the spleen after immune challenge and were 24.1 times more abundant compared with the control 24 h after injection of V. parahaemolyticus (P < 0.05). Recombinant LcRab5 protein was expressed and purified from Escherichia coli. Immunoelectron microscopy analysis showed that LcRab5 protein was localized in the mitochondria of liver and fibrous tissue of spleen. Additionally, overexpression of LcRab5 in L. crocea kidney (LCK) cells significantly enhanced tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) transcripts (P < 0.05). Our results indicate that LcRab5 is involved in the immune response of the large yellow croaker and improves the inflammatory response through activation of TNF-α and IL-6 expression.