A novel type III crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated CrusEs2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CrusEs2 was of 1237 bp, containing a 5' untranslated region (UTR) of 12 bp, a 3' UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The CrusEs2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that CrusEs2 should be classified as a type III crustin. The mRNA transcripts of CrusEs2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of CrusEs2 was cloned into pET-21a+ and expressed in Escherichia coli. The purified recombinant CrusEs2 inhibited the growth of Gram-positive bacteria at MIC of 0.093-0.37 μM. The results indicated that CrusEs2 was involved in immune response of E. sinensis against bacterial challenge.