Functional analysis of a matrix peptide involved in calcification of the exoskeleton of the kuruma prawn, Marsupenaeus japonicus

To clarify the calcification mechanism of the exoskeleton of marine crustaceans, kuruma prawns (Marsupenaeus japonicus), which are commercially important prawns in Japan, were used, and calcification-associated peptide (CAP), a matrix peptide that was previously reported in red swamp crayfish, was examined. Based on the RNA-sequencing data of kuruma prawns, a candidate Cap-1 cDNA sequence was found. Thereafter, the nucleotide sequence of kuruma prawn Cap-1 was determined by PCR methods using a specific primer set. Mature kuruma prawn CAP-1 showed a 66.7% amino acid identity with red swamp crayfish CAP-1. This sequence was presumed to be kuruma prawn CAP-1 because specific sequences, such as a chitin-binding consensus sequence, and many acidic amino acids in the C-terminal region were also observed in red swamp crayfish CAP-1. Then, a recombinant Cap-1 (rCAP-1) was expressed in Escherichia coli and purified by reverse-phase high-performance liquid chromatography. The purified rCAP-1 showed both chitin-binding and inhibitory activities of calcium carbonate precipitation. Based on Cap-1 mRNA expression analysis in the swimming legs and tail fans of juvenile prawns during molting, its mRNA expression level increased remarkably in the middle and late pre-molt stages. Cap-1-expressing cells were also identified by in situ hybridization using the swimming legs in the late pre-molt stage. It was found that kuruma prawn Cap-1 was expressed in epidermal cells under the cuticle layer. Due to knockdown using RNA interference (RNAi) methods, the Cap-1 mRNA expression in Cap-1-double-stranded RNA (dsRNA)-injected prawns decreased significantly in the middle and late pre-molt stages as compared with that in green fluorescent protein-dsRNA-injected control prawns. Furthermore, an irregularly shrunken and uneven structure appeared on the surface of the cuticle of dsRNA-Cap-1-treated prawns in the post-molt stage, and calcium deposition was inhibited by RNAi of Cap-1. Regarding marine crustaceans, to the best of our knowledge, this study is the first to determine that kuruma prawn CAP-1 has chitin-binding and anti-calcification activities and functionally changes its mRNA expression during molting. These results indicate that kuruma prawn CAP-1 plays important roles in exoskeleton calcification.