Two alpha-2 macroglobulin from Portunus trituberculatus involved in the prophenoloxidase system, phagocytosis and regulation of antimicrobial peptides.

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.