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Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana . RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three α-helices and two sets of antiparallel β-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(±0.14) A for the backbone atoms, and 1.42(±0.19) A for the heavy atoms in residues 3–105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.

The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.

Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5′- and 3′-RACE technique. The cDNA clone, named rcr gene, contained a 5′-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3′-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells.

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have con- structed bootstrap-supported phylogenetic trees that re- organize these ribonucleases into five distinct lineages— the pancreatic ribonucleases (RNases 1), the eosinophil- associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases—with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with posi- tive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contrib- uting to the rapid diversification of ribonucleases within each cluster. This pattern of evolution—rapid diversifi- cation via positive selection among sequences of a mul- tigene cluster—bears striking resemblance to what we have described for the eosinophil-associated ribonucle- ase genes of the rodent Mus musculus,a finding that may have implications with respect the physiologic function of this unique family of proteins.

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, 3JNH alpha and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel beta-sheets and three alpha-helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 alpha-protons located in the loop-1 region, an alpha 2 helix, and three beta-strands, (beta 1, beta 3 and beta 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, not in other organs. An immunohistochemical assay showed that RC-RNase was present in the regular yolk granules, but not in forming yolk granules, yolk platelets, pigment granules, mitochondria clouds or the nucleus. The RC-RNase was restricted to the lateral amorphous area of the yolk granules, and was absent from the central area that has a vitellogenin crystal lattice. The RC-RNase was extracted from yolk granules by 0.5 M NaCl and purified by dialysis and affinity chromatography. Most of the RC-RNase (94%) was found in the yolk granules, the rest RC-RNase (6%) was found in the cytosol in the form of free RNase and latent RNase. The RC-RNase extracted from yolk granules was further analyzed by immunoprecipitation and RNase activity assay on an SDS/polyacrylamide gel. Our results suggest that the RC-RNase activity is regulated by both compartmentation and inhibitor binding.

Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitate characterization of individual ribonucleases, I have developed a rapid method to separate and identify each ribonuclease from a crude sample by gel electrophoresis instead of by time-consuming purification steps. The ribonucleases in a crude sample are first separated by RNA-cast SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5′ labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed by denaturing gel electrophoresis. The base specificity of bovine pancreatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (RC-RNase), human serum ribonucleases and sweet potato leaf ribonucleases were determined by this method. Other properties of individual ribonucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.

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

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.