Abstract
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-DWD1, was first cloned from the Chinese mitten crab ( Eriocheir sinensis ). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison 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-DWD1 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-DWD1 could 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-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.
Highlights
Antimicrobial peptides are critical in the initial host defense system of many animal species [1] and exist in a wide range of organisms, including microorganisms, protozoa, invertebrates, vertebrates and plants [2]
A ClustalW2 alignment of E. sinensis DWD1 (Es-DWD1) and homologous sequences obtained from a Protein Blast search shows the conserved cysteine residues believed to be characteristic of the whey acidic protein (WAP) domain (Figure S1)
Es-DWD1 belonged to the crustin double WAP domains (DWD) group containing two WAP domains (Figure 2A)
Summary
Antimicrobial peptides are critical in the initial host defense system of many animal species [1] and exist in a wide range of organisms, including microorganisms, protozoa, invertebrates, vertebrates and plants [2]. Among the reported antimicrobial peptides, proteins containing whey acidic protein (WAP) domains, initially characterized as a milk protein, have been found in several species of both vertebrates and invertebrates [3,4]. Type I and II crustins characterized in several species of crustaceans have been shown to be abundant and to exhibit antimicrobial activity mainly against Gram-positive bacteria [3]. Two studies described crustin-like proteins in shrimp that have two WAP domains, a signal sequence and no other motifs [7,8]. These are named DWD proteins, but can be tentatively considered as type IV crustins, at least until additional information is available [9]. The synthesis of DWD proteins has been shown to be upregulated by inoculation with WSSV in M. japonicus and by injecting the heat-killed Vibrio alginolyticus in Litopenaeus vannamei in hemocytes during an early phase of infection [8,26]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
