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.     

Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.     

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.     

Zebrafish ribonucleases are bactericidal: implications for the origin of the vertebrate RNase A superfamily

Understanding the evolutionary origin of the ribonuclease (RNase) A superfamily is of great interest because the superfamily is the sole vertebrate-specific enzyme family known to date. Although mammalian RNases have a diverse array of biochemical and physiological functions, the original function of the superfamily at its birth is enigmatic. Such information may be obtained by studying basal lineages of the vertebrate phylogeny and is necessary for discerning how and why this superfamily originated. Here, we clone and characterize 3 RNase genes from the zebrafish, the most basal vertebrate examined for RNases. We report 1) that all the 3 zebrafish RNases are ribonucleolytically active, with one of them having an RNase activity comparable to that of bovine RNase A, the prototype of the superfamily; 2) that 2 zebrafish RNases have prominent expressions in adult liver and gut, whereas the 3rd is expressed in adult eye and heart; and 3) that all 3 RNases have antibacterial activities in vitro. These results, together with the presence of antibacterial and/or antiviral activities in multiple distantly related mammalian RNases, strongly suggest that the superfamily started as a host-defense mechanism in vertebrate evolution.     

The eight human “canonical” ribonucleases: Molecular diversity, catalytic properties, and special biological actions of the enzyme proteins

Human ribonucleases (RNases) are members of a large superfamily of rapidly evolving homologous proteins. Upon completion of the human genome, eight catalytically active RNases (numbered 1-8) were identified. These structurally distinct RNases, characterized by their various catalytic differences on different RNA substrates, constitute a gene family that appears to be the sole vertebrate-specific enzyme family. Apart from digestion of dietary RNA, a wide variety of biological actions, including neurotoxicity, angiogenesis, immunosuppressivity, and anti-pathogen activity, have been recently reported for almost all members of the family. Recent evolutionary studies suggest that RNases started off in vertebrates as host defence or angiogenic proteins.     

Ribonuclease A homologues of the zebrafish: polymorphism, crystal structures of two representatives and their evolutionary implications

The widespread and functionally varied members of the ribonuclease A (RNase A) superfamily provide an excellent opportunity to study evolutionary forces at work on a conserved protein scaffold. Representatives from the zebrafish are of particular interest as the evolutionary distance from non-ichthyic homologues is large. We conducted an exhaustive survey of available zebrafish DNA sequences and found significant polymorphism among its four known homologues. In an extension of previous nomenclature, the variants have been named RNases ZF-1a-c,-2a-d,-3a-e and-4. We present the first X-ray crystal structures of zebrafish ribonucleases, RNases ZF-1a and-3e at 1.35-and 1.85 Å resolution, respectively. Structure-based clustering with ten other ribonuclease structures indicates greatest similarity to mammalian angiogenins and amphibian ribonucleases, and supports the view that all present-day ribonucleases evolved from a progenitor with three disulphide bonds. In their details, the two structures are intriguing melting-pots of features present in ribonucleases from other vertebrate classes. Whereas in RNase ZF-1a the active site is obstructed by the C-terminal segment (as observed in angiogenin), in RNase ZF-3e the same region is open (as observed in more catalytically efficient homologues). The progenitor of present-day ribonucleases is more likely to have had an obstructive C terminus, and the relatively high similarity (late divergence) of RNases ZF-1 and-3 infers that the active site unblocking event has happened independently in different vertebrate lineages.     

Why ribonucleases cause death of cancer cells

Molecular properties and possible mechanisms of action of cytotoxic ribonucleases (RNases), potential antitumor therapeutics, are characterized. The analysis of recent publications and own experimental results have allowed the authors, on the one hand, to distinguish cellular components that are responsible for selective activity of exogenous RNases towards malignant cells, and on the other--to identify the contribution of definite molecular determinants to the enzyme cytotoxicity. The predominant effect of the RNase molecule charge on the cell death induction is shown. The RNase cytotoxic effects are caused by catalytic cleavage of available RNA, by products of its hydrolysis, as well as by non-catalytic electrostatic interaction of exogenous enzyme with cell components. Potential targets for RNase action in a cancer cell have been revealed. The role of modulation of the membrane calcium-dependent potassium channels and ras-oncogene functions in the RNase-induced cell damage is defined. The effect of cytotoxic RNases on gene expression via influencing the RNA interference is discussed.     

Degradation of double-stranded RNA by human pancreatic ribonuclease: crucial role of noncatalytic basic amino acid residues

Under physiological salt conditions double-stranded (ds) RNA is resistant to the action of most mammalian extracellular ribonucleases (RNases). However, some pancreatic-type RNases are able to degrade dsRNA under conditions in which the activity of bovine RNase A, the prototype of the RNase superfamily, is essentially undetectable. Human pancreatic ribonuclease (HP-RNase) is the most powerful enzyme to degrade dsRNA within the tetrapod RNase superfamily, being 500-fold more active than the orthologous bovine enzyme on this substrate. HP-RNase has basic amino acids at positions where RNase A shows instead neutral residues. We found by modeling that some of these basic charges are located on the periphery of the substrate binding site. To verify the role of these residues in the cleavage of dsRNA, we prepared four variants of HP-RNase: R4A, G38D, K102A, and the triple mutant R4A/G38D/K102A. The overall structure and active site conformation of the variants were not significantly affected by the amino acid substitutions, as deduced from CD spectra and activity on single-stranded RNA substrates. The kinetic parameters of the mutants with double-helical poly(A).poly(U) as a substrate were determined, as well as their helix-destabilizing action on a synthetic DNA substrate. The results obtained indicate that the potent activity of HP-RNase on dsRNA is related to the presence of noncatalytic basic residues which cooperatively contribute to the binding and destabilization of the double-helical RNA molecule. These data and the wide distribution of the enzyme in different organs and body fluids suggest that HP-RNase has evolved to perform both digestive and nondigestive physiological functions.     

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5′‐to‐3′ exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild‐type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5′‐to‐3′ exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.     

Structural determinants of the eosinophil cationic protein antimicrobial activity

Abstract Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.     

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.     

Endocytotic internalization as a crucial factor for the cytotoxicity of ribonucleases

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.     

Natural and engineered ribonucleases as potential cancer therapeutics

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.     

Tautomeric state of alpha-sarcin histidines. Ndelta tautomers are a common feature in the active site of extracellular microbial ribonucleases

Extracellular fungal RNases, including ribotoxins such as alpha-sarcin, constitute a family of structurally related proteins represented by RNase T1. The tautomeric preferences of the alpha-sarcin imidazole side chains have been determined by nuclear magnetic resonance and electrostatic calculations. Histidine residues at the active site, H50 and H137, adopt the Ndelta tautomer, which is less common in short peptides, as has been found for RNase T1. Comparison with tautomers predicted from crystal structures of other ribonucleases suggests that two active site histidine residues with the Ndelta tautomer are a conserved feature of microbial ribonucleases and that this is related to their ribonucleolytic function.     

Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus]

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.