The fragmentation mechanism and immune-protective effect of CfTEP in the scallop Chlamys farreri

Thioester-containing proteins (TEPs), characterized by a unique intrachain β-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in CfTEP, a previously identified thioester-containing protein in scallop Chlamys farreri, were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of CfTEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The CfTEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the CfTEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of CfTEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or β-glucan. Two bands (100 and 55 kDa) were detected using anti-CfTEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942-1472), and a single band (46 kDa) was detected by using anti-CfTEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452-496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of CfTEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum. All these results suggested that CfTEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops.