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To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase superfamily, can be classified into four different enzyme types on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and catalytic differences (action on single- and double-stranded RNAs, dependence of enzyme activity on pH, ionic strength and cations, and hydrolysis of cyclic nucleotides) have been comparatively analyzed and discussed. In addition, some data considered here concerning human nonpancreatic-type RNases may support the suggestion [Chuchillo et al. (1993) FEBS Lett. 333, 207–210] that the enzyme `ribonuclease', presently classified as `hydrolase', should be reclassified as `transferase'.© 1997 Federation of European Biochemical Societies.

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN′) and uridine and cytidine 2′, 3′-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the ‘non-secretory’ liver-type enzymes rather than to the ‘secretory’ pancreatic-type RNases.

In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.

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.

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868-3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437-3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A. Copyright (C) 2000 Federation of European Biochemical Societies.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15–18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe–NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Monomeric bovine pancreatic RNase A has been transformed into a dimeric ribonuclease with antitumor activity (Di Donato, A., Cafaro, V. and D'Alessio, G. (1994) J. Biol. Chem. 269, 17394-17396). This was accomplished by replacing the residues located in the RNase chain at positions 19, 28, 31, and 32, with proline, leucine, and two cysteine residues, respectively, i.e. those present at identical positions in the subunit of bovine seminal RNase, a dimeric RNase of the pancreatic-type superfamily, endowed with a powerful antitumor action. However, as an antitumor agent this mutant dimeric RNase A is not as powerful as seminal RNase. We report here site-directed mutagenesis experiments which have led to the identification of two other amino acid residues, glycine 38 and 111, whose substitution in the polypeptide chain of the first generation dimeric mutant of RNase A, is capable of conferring to the mutein the full cytotoxic activity characteristic of native seminal RNase. Copyright (C) 1998 Federation of European Biochemical Societies.

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40–50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.

Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA: RNA hybrids under standard assay conditions (0.15 M NaCl, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed.

A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.

Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E., and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356–363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Result indicate (1) that glycoslation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) · poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.

Evidence has been obtained that dimers and higher aggregates of pancreatic ribonuclease, already known as capable of degrading double-stranded RNAs, are also able to degrade a DNA · RNA hybrid. This finding has been discussed in the light of a hypothesis concerning the action of dimeric ribonucleases on double-stranded polyribonucleotides.

Ribonuclease A (RNase A) dimers have been recently found to be endowed with some of the special, i.e., non-catalytic biological activities of RNases, such as antitumor and aspermatogenic activities. These activities have been so far attributed to RNases which can escape the neutralizing action of the cytosolic RNase inhibitor (cRI). However, when the interactions of the two cytotoxic RNase A dimers with cRI were investigated in a quantitative fashion and at the molecular level, the dimers were found to bind cRI with high affinity and to form tight complexes. _ 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The distribution of secretory-type ribonuclease in human serum, urine and seminal plasma has been studied by immunological measurements. Inhibition of enzyme activity by antibodies against pure human seminal RNAase shows that a cross-reactive enzyme is predominant (90%) in seminal plasma and is a significant component (70–80%) in urine and serum. A competitive binding radioimmunoassay has been developed by using specific antibodies and 125 I-labelled RNAase as radioligand. The procedure, very sensitive, reproducible and specific, has been used to determine seminal RNAase levels in seminal plasma samples from 48 healthy individuals (age range, 20–58 years). The mean concentration of the enzyme was found to be 6.6 μg/ml (S.D. ± 1.9).

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The activity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013–1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride 0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.