Molecular characterization and functional analysis of the lysosomal cathepsin D-like gene in red swamp crayfish, Procambarus clarkii.

Aspartic proteinases are one of the four families of proteinase enzymes that are widely present in living organisms. They are involved in various physiological events, such as protein degradation, development, and host defense. However, the characterization and functional roles of aspartic proteinases remain to be elucidated in crustaceans. Here, we characterized a fragment of cathepsin D-like cDNA from red swamp crayfish, Procambarus clarkii (Pc-cathepsin D-like). The open reading frame of the Pc-cathepsin D-like gene contained 1152 bp, encoding a protein of 383 amino acid residues. We also evaluated the immunological role of the Pc-cathepsin D-like gene in vivo. Spatial distribution analysis revealed that the Pc-cathepsin D-like mRNA was high in the hepatopancreas, followed by the gut, gills, and hemocytes of P. clarkii. The expression levels of the Pc-cathepsin D-like gene increased following challenge with viral (polyinosinic: polycytidylic acid) and bacterial (lipopolysaccharides, peptidoglycan) PAMPs compared with PBS injection. The suppression of the Pc-cathepsin D-like gene by RNA interference significantly increased the expression of immune-associated genes. These results showed that the Pc-cathepsin D-like gene has an essential biological role in innate immune responses because it regulates the expression of immune-associated genes.

Identification and immunoregulatory role of cathepsin A in the red swamp crayfish, Procambarus clarkii.

Cathepsins are a group of lysosomal hydrolytic enzymes, broadly distributed in animals, and regulate various physiological processes. However, the immune functions of cathepsins are poorly understood in invertebrates. Therefore, to further provide information about the importance of cathepsins in the innate immune system of crustaceans, cathepsin A from Procambarus clarkii (Pc-cathepsin A) was characterized and its distribution in different tissues was determined. The immunological functions of the Pc-cathepsin A were also evaluated. The Pc-cathepsin A showed high sequence homology to cathepsins of other species, as it contained serine and histidine active sites. Quantitative RT-PCR analysis revealed that the expression of Pc-cathepsin A was highest in the gill, gut, and the hepatopancreas, with variable amounts in the muscle, stomach, heart, and hemocytes. The mRNA expression of Pc-cathepsin A was significantly increased in hepatopancreas challenged with lipopolysaccharide (LPS), peptidoglycan (PGN), and polycytidylic acid (poly I:C). The results of an in vivo analysis revealed that Pc-cathepsin A knockdown by double-stranded RNA in P. clarkii modulated the expression of immune-pathway associated genes in hepatopancreas. Collectively, these results suggest that Pc-cathepsin A modulates innate immune responses by affecting the expression of immune-pathway associated genes, thus revealing a regulatory link between Pc-cathepsin A and immune pathways in P. clarkii, and that Pc-cathepsin A plays an essential biological role in the immune defence against microbial pathogens.