Comparative sequence analysis of crustin isoform MjCRS7 and MjWFDC-like gene from kuruma shrimp Marsupenaeus japonicus shows variant of the WFDC domain.

Crustins are well known cysteine-rich cationic antimicrobial peptides (AMPs) in crustaceans that have WFDC [WAP (whey acidic protein) four-disulfide core] domain at the carboxyl terminus. Proteins containing a WFDC domain have been discovered in many invertebrates and vertebrates. Although, there have been many WFDC domain containing nucleotide sequences found in NCBI GenBank database, their distinct sequential characteristics and their role in the innate immune system is not well understood. Here, we identified a new crustin isoform from Marsupenaeus japonicus by transcriptome analysis. The full-length cDNA of this isoform (MjCRS7) consists of 537 bp that include a 489 bp open reading frame (ORF) encoding 162 deduced amino acids (aa). The sequence contains the eight conserved cysteine residues characteristic of the WFDC domain. A phylogenetic analysis showed that MjCRS7 is a type II crustin. We also identified the full-length cDNA of a M. japonicus MjWFDC-like gene. MjWFDC-like has a 543 bp ORF encoding 180 aa. In an RT-PCR analysis, MjCRS7 and MjWFDC-like transcripts were mainly detected in gill tissue. An alignment of MjCRS7 and MjWFDC-like with previously reported M. japonicus crustin isoform 1-5 (MjCRS1-5) showed variation in the WFDC-like domain. Neither of the genes was responsive to Vibrio parahaemolyticus, Vibrio penaeicida or white spot syndrome virus (WSSV) either by immersion or injection challenge test. Although crustins are mainly antimicrobial peptides, the present results suggest that MjCRS7 may have other roles in M. japonicus.

Gills specific type 2 crustin isoforms: Its molecular cloning and characterization from kuruma shrimp Marsupenaeus japonicus.

Crustins are diverse group of antimicrobial peptides (AMPs) that have numerous isoforms mainly identified from hemocytes in decapods crustacean. However, little is known about its presence solely in gills tissue. In this study, we found two new crustin isoforms MjCRS8 and MjCRS9 by using transcriptome analysis from gills. Open reading frame of MjCRS8 and MjCRS9 were 593 bp and 459 bp encoding 197aa and 152aa, respectively. Tissue distribution analysis indicated that both MjCRS8 and MjCRS9 are expressed only in gills tissue. Multiple sequence alignment and phylogenetic analysis with previously reported crustin suggested that both MjCRS8 and MjCRS9 belong to type 2 crustin family. Experimental infection was conducted against Vibrio parahaemolyticus and white spot syndrome virus (WSSV) by immersion test. However, no significant upregulation was observed.