Biochemical Characterization of Ribonuclease Activities From the Sea Cucumber Eupentacta fraudatrix

Background: The sea cucumber Eupentacta fraudatrix is a highly interesting model organism for research due to the remarkable ability of the organism to regenerate lost organs following evisceration. In animals after evisceration, increased expression levels of genes such as Sox (SRY-related HMG-box, transcription factors), MMP (matrix metalloproteinases), TIMP (tissue inhibitors of metalloproteinases), and PIWI (ribonuclease-active proteins) have been observed. Since ribonucleases release nucleotides for the synthesis of new molecules and regulate cellular processes by acting on various RNA species, these enzymes are hypothesized to play a significant role in regeneration. Methods: Holothurian homogenate was fractionated chromatographically into several protein fractions. Ribonuclease activity was analyzed using a specific microRNA (miRNA) as a substrate, as well as by an in-gel activity assay. Results: This study examined the ribonuclease activity of E. fraudatrix proteins precipitated with ammonium sulfate from whole-organism homogenates, focusing on pH dependence and substrate specificity. The optimal pH for hydrolyzing a specific miRNA related to the sea cucumber Apostichopus japonicus and a non-specific homo-oligonucleotide, (pA)23, was 5.0 and 6.0, respectively. Inhibition of ribonuclease activity in the presence of EDTA (ethylenediaminetetraacetic acid), Mg2+, and Ca2+ indicates dependence on metal ions. Hydrolysis of the specific substrate was about 25% higher than that of the non-specific substrate and produced five cleavage sites. Using in-gel activity analysis, five protein bands with ribonuclease activity were detected in E. fraudatrix homogenate, with apparent molecular masses ranging from 15 to 77 kDa. Conclusions: This work represents the first report describing the biochemical properties of a ribonuclease from this holothurian species. Five protein forms with ribonuclease activity, differing in electrophoretic mobility, were identified. The biochemical characteristics of these enzymes resemble those of ribonucleases from other echinoderms. Ribonucleases from echinoderms are promising tools for molecular biology applications, including DNA purification, RNA analysis and mapping, as well as for biomedicine, particularly in the development of antiviral agents.