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

The non-specific protease inhibitor alpha-2-macroglobulin (A 2 M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A 2 M has been developed in echinoderms to date. In this study, the full-length cDNA of A 2 M 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 HaA 2 M . HaA 2 M possesses all three conserved functional domains of known A 2 M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A 2 Ms, the histidine residue from the catalytical regions is well conserved in HaA 2 M. HaA 2 M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A 2 M 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 HaA 2 M resulted in a significant reduction of HaA 2 M gene transcript level (86%). RNAi-mediated silencing of HaA 2 M gene significantly decreased the A 2 M 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 HaA 2 M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra .