Cloning and characterization of the target protein subunit lst8 of rapamycin in Apostichopus japonicus

Abstract

Autophagy plays an important role in the immune defense systems of vertebrates through the interaction between the lethal with SEC13 protein 8 (lst8) and the mechanistic target of rapamycin. In the present study, a novel invertebrate lst8 homologue is identified from Apostichopus japonicus (designated as Ajlst8) via polymerase chain reaction. The full-length complementary DNA of Ajlst8 comprises a 5ʹ-untranslated region (UTR) of 78 base pair (bp), a 3ʹ-UTR of 479 bp, and a putative open reading frame of 951 bp; hence, 316 amino acids are encoded. Structural analysis shows that the deduced amino acid of Ajlst8 shares six typical WD40 domains (28 aa–248 aa). Spatial expression analysis indicates that Ajlst8 is ubiquitously expressed in all the examined tissues, with a larger magnitude in coelomocytes. Vibrio splendidus infection in vivo and lipopolysaccharide exposure in vitro can significantly upregulate the messenger RNA expression of Ajlst8 by 2.39-fold and 1.93-fold compared with the control group, respectively. LPS exposure could also significantly induced the protein level of Ajlst8 to 2.38-fold and the autophagy level was markedly increased by 3.08-fold under same condition. The RNA interference of Ajlst8 in primary coelomocytes also reduces the relative expression of autophagy with a 0.71-fold decrease in the ratio of LC3-II/LC3-I compared with that in the control group. These results indicate that Ajlst8 is a novel immune regulator that may be involved in the antibacterial response process of sea cucumber by regulating autophagy.