Characterization and the role of α2-macroglobulin from Macrobrachium nipponense in response to ammonia-N stress

Abstract

Ammonia-N is a major physiological challenge for prawns in culture. The alpha-2-macroglobulin (α2-macroglobulin, α2M) gene is a multifunctional protein with a significant role in the innate immunity. In this study, the full-length cDNA of α2M (Mnα2M) from M. nipponense was amplified and the 4805 bp cDNA contains a 4422 bp open reading frame (ORF). This cDNA encodes a polypeptide comprising 1473 amino acids, with a predicted molecular weight of 163.45 kDa. The sequence of Mnα2M contains three putative conserved functional domains including a bait region, a thiol ester domain and a receptor binding domain which are present in other invertebrate and vertebrate α2Ms. Multiple alignment analysis showed that α2M in M. nipponense and other crustaceans shared high similarity. Severe histological alterations were observed in M. nipponense hepatopancreas. Quantitative real-time PCR (qRT-PCR) confirmed that Mnα2M was expressed in all examined tissues, with the high expression level in the hepatopancreas, hemocyte and gills. The expression of Mnα2M in hepatopancreas, hemocyte and gills was significant up-regulated at 12 h, 24 h and 48 h under ammonia-N stress. Western blot analysis further revealed that the Mnα2M protein was distributed in all tissues, with the high expression level in hepatopancreas, hemocyte and gills, which was consistent with the qRT-PCR results. These results suggest that high concentrations of ammonia-N has significant effects on the M. nipponense and Mnα2M might be involved in the immune response to ammonia-N stress.