1H, 13C and 15N resonance assignments and secondary structure of the cytotoxic protein RNase 4 from bullfrog Rana catesbeiana oocytes.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs, and share sequence similarity with each other. They all belong to the RNase A superfamily but possess different cytotoxicities, base specificities and ribonucleolytic activities. RC-RNase 4, one of the five novel ribonucleases isolated from the oocytes of Rana catesbeiana, is a 106 amino acid protein with a pyroglutamate residue at the N-terminus (Liao et al., 2000). Phylogenetic analysis of RNase A superfamily reveals that RC-RNase 4 is very similar to onconase, a cytotoxic ribonuclease isolated from the oocytes of bullfrog Rana pipiens, which is now undergoing phase III clinical trials for cancer treatment (Juan et al., 1998). RC-RNase 4 shares 66.0% sequence identity with and its cytotoxicity resembles that of onconase. However, RC-RNase 4 has the base specificity of CpG whereas onconase has that of UpG. Thus, it is interesting to investigate the possibility of RC-RNase 4 as a potential agent for tumor therapy and to study its structural–functional relationship. In addition, among the newly identified frog ribonucleases, only RC-RNase 4 shows distinct CD data, which display two negative ellipticities at 212 and 229 nm when compared to one minimum (∼ 211 nm) observed for the other frog ribonucleases. The melting studies also reveal that RC-RNase 4 possesses higher thermostability. To elucidate RC-RNase 4’s similarities to onconase and its unique properties, we applied NMR