Molecular characterization of two newly identified glycine rich crustin genes (Mn-Gly-Cru3, Mn-Gly-Cru4) and their function in Macrobrachium nipponense innate immunity

Abstract

Crustin, as an important antibacterial peptide in crustacean, plays a role in the defense against the invasion of foreign pathogens in the innate immune system. In this study, two glycine rich crustin genes (named as Mn-Gly-Cru3 and Mn-Gly-Cru4) were cloned in Macrobrachium nipponense. The full-length cDNA of Mn-Gly-Cru3 and Mn-Gly-Cru4 were 740 and 781 bp with open reading frames of 525 and 576 bp that encode 174 and 191 amino acids, respectively. The genome structure of Mn-Gly-Cru3 and Mn-Gly-Cru4 shows that both contain two exons and one intron. Both Mn-Gly-Cru3 and Mn-Gly-Cru4 protein contains a signal peptide, a cysteine-rich region, a glycine-rich region, and a whey acidic protein domain. Evolutionary analysis showed that Mn-Gly-Cru1, Mn-Gly-Cru2, Mn-Gly-Cru3 and Mn-Gly-Cru4 were clustered together. Mn-Gly-Cru3 and Mn-Gly-Cru4 were widely distributed in multiple tissues. After white spot syndrome virus (WSSV), Staphylococcus aureus or Vibrio parahaemolyticus challenge, the expression levels of Mn-Gly-Cru3 and Mn-Gly-Cru4 in the hemocytes, gills, and stomach were significantly changed. Mn-Gly-Cru3 and Mn-Gly-Cru4 knockdown remarkably increased the copy number of WSSV and the number of S. aureus and V. parahaemolyticus colonies in vivo. Purified rMn-Gly-Cru3 and rMn-Gly-Cru4 proteins could bind to a variety of bacteria and they also had binding activity toward LPS or PGN. Furthermore, rMn-Gly-Cru4 had a stronger binding activity to polysaccharides than rMn-Gly-Cru3. Our study indicates that Mn-Gly-Cru3 and Mn-Gly-Cru4 in M. nipponense are involved in the innate immune defense against bacteria or viruses.