Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells

Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems.     

Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.     

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.     

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

Rapid Diversification of RNase A Superfamily Ribonucleases from the Bullfrog, Rana catesbeiana

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 constructed bootstrap-supported phylogenetic trees that reorganize 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 positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.     

Enzymatic incorporation into oligonucleotides of modified nucleosides

Behaviour of modified nucleosides, tRNA components, and their analogues has been studied in the internucleotide bond formation catalysed by ribonucleases of various substrate specificity, polynucleotide phosphorylases, and T4 RNA ligase and the results are summarised in this paper. Pseudouridine, dihydrouridine, ribothymidine, 5-methylcytidine, inosine, and 6-methyladenosine can participate in the reaction of internucleotide bond formation the presence of most ribonucleases used, viz. Pb2, Pcl2, Pb1, Pch1, C2, T1, pancreatic RNase. 3-Methylcytidine and 4-acetylcytidine form internucleotide bond (as phosphate acceptors) usually by means of guanyl-specific ribonucleases, whereas 1-methylandenosine is incorporated with ribonuclease Pel2. 7-Methylguanosine and 1-methylguynosine 2',3'-cyclophosphates can be used as phosphate donors in the presence of ribonuclease Pb2; in the similar enzymatic reaction 6-isopentenyladenosine is an uneffective acceptor.     

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.     

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.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.     

Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells

The primary structure of an extracellular ribonuclease (RNase LE) from Pi-depleted media of cultured cells of Lycopersicon esculentum L. cv. Lukullus has been determined. This was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the reduced and S-ethylpyridylated protein. RNase LE consists of 205 amino acid residues and has a molecular mass of 22,666 Da and an isoelectric point of 4.24. The enzyme contains 10 half-cystines. There are no potential N-glycosylation sites in the sequence. The sequence of RNase LE is homologous with those of self-incompatibility proteins of several higher plant species and with those of a number of fungal RNases. The sequence similarity with the family of self-incompatibility proteins is greater than with the fungal RNases, suggesting that the self-incompatibility proteins arose from ancestral RNase by gene duplication after the divergence of higher plants and fungi. Two pentapeptide sequences, i.e. HGLWP and KHGTC (or KHGSC), are present at identical positions in all the aligned proteins, suggesting that they contribute to the active site.     

Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans

The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H₂O₂ challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiating TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicate that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response.Dedicated to Dr Joseph Elmer Varner.     

Ribonuclease from Alcaligenes eutrophus: Activation by heat treatment,partial purification and some properties

Summary Heat treatment (10 min at 65° C) of cells of Alcaligenes eutrophus, which resulted in the release of RNA degradadation products into the medium, was found to activate cellular ribonucleases. Two ribonucleases degrading yeast RNA were found, one localized in the periplasmic space and the other in the soluble fraction of the ribosomes. Compared to non-heated cells, in the heat-treated cells the former enzyme, the cell debris ribonuclease, was present at an eightfold increased specific activity, and the latter, the cytoplasmic ribonuclease, was present at a fourfold increased specific activity. This increase was due to the inactivation of a thermolabile inhibitor and to denaturation of part of the soluble protein during heat treatment. With respect to their properties the enzymes were similar; they had endonuclease activity and hydrolysed only RNA. They were heat-stable, resistent to trypsin, highly sensitive to a ribonuclease inhibitor isolated from the same bacterium and were partially inhibited by ATP and GTP. These properties provided a partial explanation for the mechanism of the release of RNA dagradation products from A. eutrophus cells after heat treatment.Abbreviations DNA Deoxyribonucleic acid - RNA Ribonucleic acid - tRNA Transfer RNA - DNase Deoxyribonuclease - RNase Ribonuclease - d-RNase debris RNase - c-RNase cytoplasmic RNase     

The role of the promoter and leader sequences of extracellular ribonuclease from Bacillus amyloliquefaciens in regulation of enzyme biosynthesis

It is shown that in the medium rich with inorganic phosphate there is a stimulation of biosynthesis of ribonuclease from B. amyloliquefaciens (barnase) by actinomycin D, while biosynthesis of ribonucleases from B. intermedius (binase) and B. pumilus (KNase Bpu) in these conditions was suppressed. Features of biosynthesis of binase and RNase Bpu, directed by the barnase promoter, and also expression of chimeric gene of RNase Bpu with leader peptide of barnase were investigated. It was established that stimulation of synthesis of extracellular ribonuclease from B. amyloliquefaciens in the presence of actinomycin D was defined by structure of leader sequences.     

Cytotoxic ribonucleases: the dichotomy of Coulombic forces

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.     

Determination of base specificity of multiple ribonucleases from crude samples

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.Abbreviations RNase ribonuclease - SDS sodium dodecyl sulfate     

Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T₂ ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.