A Novel Cationic Ribonuclease with Antimicrobial Activity from Rana dybowskii

A novel ribonuclease (RNase) A superfamily gene (Rdronc) has been cloned from the frog Rana dybowskii. The deduced amino acid sequence shows that it belongs to the ribonuclease A superfamily, with the highest identity, 73%, to Rana pipiens onconase. Adaptive evolution analysis based on maximum likelihood models of codon substitution has been conducted on 10 members of the Rana RNases of subcluster B. Rapid adaptive evolution and multiple positive selection sites have been detected, which indicates that these genes may be evolving under positive selection pressure. Functional assay demonstrates that the recombinant Rdronc protein possesses antimicrobial activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa and weaker antimicrobial activity against Gram-positive Staphylococcus aureus and yeast Candida albicans. Our findings support the hypothesis that ribonuclease A superfamily members may function in host defense of early-diversified vertebrates.     

Onconase: an unusually stable protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.     

Contribution of chain termini to the conformational stability and biological activity of onconase

Onconase, a member of the RNase A superfamily, is a potent antitumor agent which is undergoing phase III clinical trials as an antitumor drug. We have recently shown that onconase is an unusually stable protein. Furthermore, the protein is resistant to the action of proteases, which could influence its use as a drug, prolonging its biological life, and leading to its renal toxicity. Our investigation focused on the contribution of chain termini to onconase conformational stability and biological activities. We used differential scanning calorimetry, isothermal unfolding experiments, limited proteolysis, and catalytic and antitumor activity determinations to investigate the effect of the elimination of the two blocks at the chain termini, the N-terminal cyclized glutamine and the C-terminal disulfide bridge between the terminal Cys104 and Cys87. The determination of the thermodynamic parameters of the protein led to the conclusion that the two blocks at onconase chain termini are responsible for the unusual stability of the protein. Moreover, the reduced stability of the onconase mutants does not influence greatly their catalytic and antitumor activities. Thus, our data would suggest that an onconase-based drug, with a decreased toxicity, could be obtained through the use of less stable onconase variants.     

Structural-functional study of recombinant forms of onconase

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in nondenaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure-function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.     

松鼠胰腺型核糖核酸酶A基因的克隆与表达

核糖核酸酶A(ribonucleases A,RNases A)构成一个大家族,其成员表现出不同程度的水解RNA的酶活性。一些成员还表现出特殊的生物学活性。旨在克隆松鼠核糖核酸酶A的基因,利用原核细胞系统表达重组蛋白,并进行初步的酶学性质的研究。结果表明,所克隆的基因属于核糖核酸酶A家族,保留了核糖核酸酶A家族酶活性必要的保守序列。进化分析表明,该基因属于松鼠胰腺型核糖核酸酶基因,与其它啮齿类动物胰腺型酶一样,重组的核糖核酸酶具有酶活性,为进一步进行啮齿类动物核糖核酸酶的宿主防卫功能研究打下了基础。     

Thermal Stability of Onconase and Some Mutant Forms

Onconase is a small globular protein of the pancreatic ribonuclease superfamily. It is an important molecule because it possesses a selective antitumor activity. The interesting finding is that onconase has a high thermal stability, with a denaturation temperature close to 90d`C at p^H 6.0. A detailed comparison between the tertiary structures of onconase and bovine pancreatic ribonuclease has been accomplished in order to identify the molecular determinants of the high stability. The results of differential scanning calorimetry measurements confirm that the mutant forms of onconase, designed to be less stable than the parent enzyme, exhibit lower denaturation temperatures. In particular, the disulfide bridge at the C-terminus of onconase seems to play a pivotal role in stability.     

Structure–Function Study of Recombinant Onconase Variants

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in non-denaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure–function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.     

Solution structure of the cytotoxic RNase 4 from oocytes of bullfrog Rana catesbeiana

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 alpha-helices and two sets of antiparallel beta-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.     

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.     

Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Transfer RNA cleavages by onconase reveal unusual cleavage sites

Onconase, a protein from amphibian eggs and a homologue of pancreatic ribonuclease (RNase) superfamily, is cytotoxic, exhibits antitumor and antiviral activity, and is in phase III clinical trials. It has been shown to predominantly target cellular tRNA on its entry into mammalian cells (Saxena, S. K., Sirdeshmukh, R., Ardelt, W., Mikulski, S. M., Shogen, K., and Youle, R. J. (2002) J. Biol. Chem. 277, 15142-15146). Cleavage site mapping using natural tRNA substrates, in vitro, revealed predominant cleavage sites at UG and GG residues. Cleavages at UG or the less intense cleavages at CG sites are consistent with the known base specificity of onconase. However, predominance of cleavages at selected G-G bonds is unusual for a homologue of pancreatic RNases. Interestingly, in at least three of the four tRNA substrates studied, the predominant cleavages mapped in the triplet UGG located in the context of the variable loop or the D-arm of the tRNA. The cleavage specificity of onconase observed by us thus indicates another special feature of this enzyme, which may be relevant to its cellular actions.     

RNase 8, a novel RNase A superfamily ribonuclease expressed uniquely in placenta

We report the identification and characterization of the gene encoding the eighth and final human ribonuclease (RNase) of the highly diversified RNase A superfamily. The RNase 8 gene is linked to seven other RNase A superfamily genes on chromosome 14. It is expressed prominently in the placenta, but is not detected in any other tissues examined. Phylogenetic analysis suggests that RNase 7 is the closest relative of RNase 8 and that the pair likely resulted from a recent gene duplication event in primates. Further analysis reveals that the RNase 8 gene has incorporated non-silent mutations at an elevated rate (1.3 × 10^–9 substitutions/site/year) and that orthologous RNase 8 genes from 6 of 10 primate species examined have been deactivated by frameshifting deletions or point mutations at crucial structural or catalytic residues. The ribonucleolytic activity of recombinant human RNase 8 is among the lowest of members of this superfamily and it exhibits neither antiviral nor antibacterial activities characteristic of some other RNase A ribonucleases. The rapid evolution, species-limited deactivation and tissue-specific expression of RNase 8 suggest a unique physiological function and reiterates the evolutionary plasticity of the RNase A superfamily.     

Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.     

The success of the RNase scaffold in the advance of biosciences and in evolution

In 1938 the new word "ribonuclease" was coined to name an enzyme capable of degrading RNA, before the name "ribonucleic acid" was accepted, as at that time RNA was still labeled YNA, for Yeast Nucleic Acid. Later, four Nobel prizes were awarded to investigators working with the "ribonuclease", RNase A from bovine pancreas. Their work greatly advanced our knowledge of protein chemistry and biology, by producing the first complete amino acid composition and the first covalent structure of a protein, the first complete synthesis of an enzyme, and the discovery that the three-dimensional structure of a protein is dictated by its amino acid sequence. Today, well over 100 homologs of RNase A have been identified in all tetrapods, and recently in fishes. Based on the latter findings, a vertebrate RNase superfamily has been appropriately defined, with RNase A as its prototype. Thus, the success of the RNase structure and function not only in promoting the advance of biosciences, but also in evolution, has become clear. Several RNases from the superfamily are endowed with non-catalytic "special" bioactions. Among these are angiogenins, characterized by their ability to stimulate the formation of blood vessels. Recently, four RNases have been identified in Danio rerio, or zebrafish, produced as recombinant proteins, and characterized. As two of them have angiogenic activity, the hypothesis is made that the whole superfamily of vertebrate RNases evolved from early angiogenic RNases. Given the microbicidal activity of some mammalian angiogenins, and of the reported fish angiogenins, the alternative hypothesis is also discussed, that the ancestral RNases were host-defense RNases.     

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine–lysine– histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3’s cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.     

Correlation of folding kinetics with the number and isomerization states of prolines in three homologous proteins of the RNase family

Several studies attribute the slower phases in protein folding to prolyl isomerizations, and several others do not. A correlation exists between the number of prolines in a protein and the complexity of the mechanism with which it folds. In this study, we have demonstrated a direct correlation between the number of cis-prolyl bonds in a native protein and the complexity with which it folds via slower phases by studying the folding of three structurally homologous proteins of the ribonuclease family, namely RNase A, onconase and angiogenin, which differ in the number and isomerization states of their proline residues.     

X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity

Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.     

Ribonucleases and angiogenins from fish

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities.