Cloning and functional analysis of a pacifastin-like protein from the sea cucumber, Apostichopus japonicus

Abstract

Pacifastin family proteins play a crucial role in regulating innate immune responses such as phagocytosis in invertebrates. However, the function of the Ajpacifastin-like counterpart in the sea cucumber Apostichopus japonicus remains elusive. In this study, the pacifastin gene of A. japonicus was cloned, characterized and named Ajpacifastin-like. The open reading frame of Ajpacifastin-like is 1497 bp in length and encodes a polypeptide containing 498 amino acid residues. Structural analysis revealed that the protein encoded by Ajpacifastin-like contains two pacifastin light chain domains (amino acids 287–322 and amino acids 376–407). Real-time reverse transcriptase PCR showed that Ajpacifastin-like mRNA is ubiquitously expressed in all tissues examined, with the highest expression in muscle. Ajpacifastin-like mRNA expression was significantly upregulated to 3.27-fold after challenge with Vibrio splendidus for 24 h. To explore the function of the Ajpacifastin-like protein in the immune response of A. japonicus, dsRNA interference with Ajpacifastin-like expression and with the expression of its postulated target gene was performed. Flow cytometry analysis showed that the rate of phagocytosis by coelomocytes increased to 1.21-fold in individuals treated with specific Ajpacifastin-like siRNA. However, rate of phagocytosis by coelomocytes decreased to 86% in individuals treated with Ajphenoloxidase siRNA. These results show that the Ajpacifastin-like gene is ubiquitously expressed in almost all tissues and that Ajpacifastin-like protein acts as an immunomodulatory factor via phenoloxidase to mediate phagocytosis by coelomocytes in pathogen-challenged A. japonicus.