Salvatore Sorrentino

Structure–function relationships in human ribonucleases: main distinctive features of the major RNase types

© 1997 Federation of European Biochemical Societies.

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.

Revisiting the action of bovine ribonuclease A and pancreatic-type ribonucleases 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.

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP)

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.

Interactions of the cytotoxic RNase A dimers with the cytosolic ribonuclease inhibitor

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.

The two dimeric forms of RNase A

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.

A ribonuclease from human seminal plasma active on double -stranded RNA

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.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action

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.

Ionic control of enzymic degradation of double-stranded RNA

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 acstivity 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.

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

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.