Molecular characterization and expression of two types of crustin-like proteins in the Morotoge shrimp, Pandalopsis japonica

Abstract

Crustins are cysteine-rich cationic antimicrobial peptides (AMPs) found in decapod crustaceans. Total nine crustin genes (Paj-CrusIa, Ib, Ic, Id, Ie, If, IIa, IIb and IIc) were identified in the morotoge shrimp, Pandalopsis japonica as result of bioinformatic analysis and PCR-based cloning strategy. Deduced amino acid sequences of isolated Paj-Crus genes ranged from 99 to 178 amino acid residues (10.6–17.8 kDa). Sequence analysis of nine isolated Paj-Crus genes and 100 different crustins from various decapod crustaceans revealed that a splice site and KXXXCP motif within the WAP domain may be the main criteria for classifying type I and II crustins, suggesting that the two types of crustin genes may have been generated by different processes. We also identified three intron-less crustin I genes (Paj-Crus Id, Ie and If) for the first time, which may have been generated by gene duplication. The tissue distribution profiles showed that Paj-CrusI genes were expressed predominantly in the gill and epidermis, whereas Paj-CrusII genes were expressed ubiquitously, suggesting that the two types of crustins may play different roles in various tissues or under different physiological conditions. In order to know the functional differences between two types of crustins, four recombinant crustins (Paj-CrusIa, Ib, IIa, IIc) were expressed in E. coli and then purified with his-tag column. All four purified recombinant crustins showed no antimicrobial activity against both Gram-positive and Gram-negative bacteria.