Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA (original) (raw)
Related papers
Origin of the Catalytic Activity of Bovine Seminal Ribonuclease against Double-Stranded RNA †
Biochemistry, 1998
Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.
Degradation of double-stranded RNA by a monomeric derivative of ribonuclease BS-1
Biochimica et biophysica acta, 1975
Double-stranded RNA, resistant to the action of pancreatic monomeric RNAase A, is actively degraded by seminal dimeric RNAase BS-1. Evidence is presented that a monomeric derivative of seminal RNAase degrades double-stranded RNA as efficiently as the parent dimeric molecule. This finding is discussed in the light of the hypothesis previously advanced that two active sites simultaneously available on an enzyme molecule may be responsible for degradation of double-stranded polyribonucleotides.
Dimeric structure of seminal ribonuclease
FEBS Letters, 1972
RNAase BS-l., the major component of ribonuclease activity in bull seminal plasma, is a basic protein with a molecular weight of 29,000 [ 11. Its catalytic properties are very similar to those of bovine pancreatic RNAase A [2] , except for a lower kcat and the ability to degrade also double-stranded RNA under conditions in which RNAase A is only slightly active [3]. We wish to report on the subunit structure of this enzyme, the first instance, to our knowledge, of a dimeric ribonuclease. This conclusion rests on several lines of evidence: i) peptide mapping of the protein; ii) quantitation of end groups; iii) estimation of the subunit molecular weight by electrophoresis on polyacrylamide gels in sodium dodecylsulphate; iv) estimation of the number of covalently linked species obtained by amidination of the protein with a crosslinking reagent. The peptide map of RNAase BS-1 was obtained by tryptic digestion of the protein oxidized with performic acid [4]. A total of 19 f 2 spots was obtained in several experiments (fig. la). From the ammo acid composition (28 lysine and 8 arginine residues per molecule), one would expect a number of about 37 peptides for a protein consisting of a single polypeptide chain. The observed value can therefore only be explained if one assumes that the protein is made up of two identical, or very similar, subunits. On the other hand, the possibility of RNAase BS-1 being a dimer of RNAase A is ruled out by a comparison of the peptide maps of the two proteins (fig. la and b). Several peptides appear to be different, and this is in * This is paper no. 3 on Bull Semen Ribonucleases. The first two papers have been published elsewhere [ 1,2].
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1989
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 125I-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 micrograms/ml (S.D. +/- 1.9).
Ionic control of enzymic degradation of double-stranded RNA
Biochimica Et Biophysica Acta (bba) - Nucleic Acids and Protein Synthesis, 1980
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.
The dual-mode quaternary structure of seminal RNase
Proceedings of the National Academy of Sciences, 1992
Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells. Bovine seminal ribonuclease (BS-RNase) is a homodimeric ribonuclease-in fact, the only dimeric RNase isolated thus far. Its enzymic properties are unusual for a ribonuclease, as it cleaves effectively singleand double-stranded RNA and is allosterically regulated in the rate-determining step of the reaction. These properties are matched by its unusual biological, noncatalytic actions, including its selective toxicity toward tumor cells, activated T cells, and the male germ cell line (ref. 1 and references cited therein). In the structure of naturally dimeric BS-RNase determined by x-ray crystallography (2, 3), the two subunits interchange their N-terminal segments (see Fig. 1A) as do the monomers of artificially dimerized bovin' pancreatic RNase (RNase A) in the structure proposed by Crestfield and others (4, 5). A
In vitro studies on selective inhibition of tumor cell growth by seminal ribonuclease
Cancer research, 1980
The effect on cell growth of bovine seminal RNase has been tested on cells cultured in vitro. A selective inhibition of growth has been observed on tumor cells as compared to normal cells using several virus-transformed cell lines and a neuroblastoma line. The different cellular response of virus-transformed cells does not appear to depend on a differential permeability to the protein of transformed cells with respect to nontransformed ones. The selective cytotoxic action of seminal RNase on tumor cell growth was also compared with the action of other structurally related RNases and of various RNase derivatives prepared by specific chemical modifications. The results indicate that the protein dimeric structure and its enzymic activity are essential requirements for its action.
FEBS Letters, 2013
Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111-116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.
Molecular and Cellular Biochemistry, 1992
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 NaC1, 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. (Mol Cell Biochem 117: 139-151, 1992) Key words." double-stranded RNA, ribonuclease(s), bovine RNase A, pancreatic-type RNases, nucleic acid helixdestabilizing proteins Abbreviations: RNase -Ribonuclease, dsRNA -Double-stranded RNA, ssRNA -Single-stranded RNA, poly(A) : poly(U), poly(I) : poly(C) -Double-stranded Homopolymers formed between Polyadenylate and Polyurydilate, and Polyinosinate and Polycytidylate, respectively, poly(dA) : poly (rU) -Double-stranded complex formed between Polydeoxyriboadenylate and Polyribouridylate, poly(A), poly(C) -Polyadenylate and Polycytidylate, respectively, poly[d(A-T)] -Double-stranded Homopolymers formed between Polydeoxyriboadenilate and Polydeoxyribothymidylate, poly(dA-dT) : poly (dA-dT) -Double-stranded alternating copolymers, SSC -0.15 M Sodium Chloride, 0.015 M Sodium Citrate pH 7
FEBS Journal, 2010
Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity. Abbreviations BS-RNase, bovine seminal ribonuclease; DSC, differential scanning calorimetry; GSH, glutathione; hA-BS-RNase, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; hA-mBS, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; mBS, monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; MxM, dimeric form of bovine seminal ribonuclease in which the chains swap their N-termini; M=M, unswapped dimer of bovine seminal ribonuclease; NCD, noncovalent dimer; PDB, Protein Data Bank; RI, ribonuclease inhibitor; RNase A, bovine pancreatic ribonuclease; S 80 -BS-RNase, R80S variant of bovine seminal ribonuclease; S 80 -hA-BS-RNase, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; S 80 -hA-mBS, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; S 80 -mBS, R80S variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties.