Expression And Function Study Of A Double WAP Domain-containing Protein Es-DWD1from Eriocheir Sinensis Exhibits Antimicrobial Activities

As all important crustacean, Eriocheir sinensis is good experiment materials which can be used to research the innate immune system of invertebrate. By researching the interrelated genes in the innate immune system, we call deeply know the immune principle in the antibacterial and antiviral responses in crustacean, which can provide new strategies for the disease control in the crab aquaculture. These researches can also provide data for understanding the innate immune system of invertebrate.Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWDl, was first cloned from the Chinese mitten crab (Eriocheir sinensis). The full-length Es-DWD1cDNA was1193bp, including a411bp open reading frame (ORF) encoding136amino acids with a signal peptide of22amino acids in the N-terminus. A comparision with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWDl transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1(rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1could exert agglutination activities against Bacillus subtilis and Pichia pastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.