A novel type III crustin (Crus Es2) identified from Chinese mitten crab Eriocheir sinensis

Abstract

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated Crus Es2) 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 Crus Es2 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 Crus Es2 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 Crus Es2 should be classified as a type III crustin. The mRNA transcripts of Crus Es2 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 Crus Es2 was cloned into pET-21a (+) and expressed in Escherichia coli. The purified recombinant Crus Es2 inhibited the growth of Gram-positive bacteria at MIC of 0.093–0.37 μM. The results indicated that Crus Es2 was involved in immune response of E. sinensis against bacterial challenge.