Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra

• The full-length cDNA of alpha-2-macroglobulin (A2M) was obtained from the sea cucumber Holothuria atra.
• Higher expression level of HaA2M was found in the body wall and coelomocytes, which are involved in body wall and cellular response, respectively.
• The expression of HaA2M in immune tissues by treatment with V. alginolyticus and Poly (I: C) indicated that HaA2M is an immune-related gene.
• The changes in HaA2M activity by treatment with V. alginolyticus and Poly (I: C) indicated that A2M is an immune-related protein.
• In this study, putative differences in the HaA2M mRNA and A2M activity levels were investigated in H. atra.
• The gene silencing of HaA2M suggested that HaA2M is an essential molecule in the innate defense against V. alginolyticus infections.

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.