Human Placental Ribonuclease Inhibitor Research Articles

Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis

The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis. Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay. Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition. The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40. The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined. While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine. Both substitutions substantially decreased angiogenic activity. The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site. The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.

Primary structure of human placental ribonuclease inhibitor [Erratum to document cited in CA109(23):207334b

Primary structure of human placental ribonuclease inhibitor [Erratum to document cited in CA109(23):207334b

Expression of human placental ribonuclease inhibitor in Escherichia coli

Human placental ribonuclease inhibitor (PRI) has been expressed in and isolated from Escherichia coli . Its apparent molecular weight, immunoreactivity and amino acid composition are virtually identical with those of native PRI. It inhibits the enzymatic activities of either angiogenin, a blood vessel inducing protein homologous to pancreatic RNase (RNase A), or RNase A in a stoichiometry of 1:1. Recombinant PRI binds to angiogenin and RNase A with K i values of 2.9 × 10 −16 M and 6.8 × 10 −14 M, respectively, comparable to the affinities of native PRI for these enzymes. Thus, these results confirm that PRI inhibits angiogenin more effectively than RNase A.

Primary structure of human placental ribonuclease inhibitor.

The primary structure of the human placental ribonuclease inhibitor (PRI), a tight-binding inhibitor of angiogenin and pancreatic ribonucleases, has been determined from the cDNA. The sequence of the mature protein is composed of 460 amino acids, yielding a molecular mass of 49,847 g/mol. Peptides comprising 92% of the predicted sequence were isolated from a tryptic digest of PRI, and direct sequence information obtained for 65% of the molecule agreed at all positions with the sequence predicted from the cDNA. The amino acid sequence of PRI contains seven direct internal repeat units, each 57 amino acids in length. These repeat units comprise 87% of the molecule. The average degree of identity between any two is 39%. A region within each repeat unit displays similarity to tandem, leucine-rich repeats found in six other proteins. Modification of PRI with iodoacetic acid, p-(hydroxymercuri)benzoate, and 5,5'-dithiobis(2-nitrobenzoic acid) reveals that at least 30 of the 32 cysteine residues of PRI are in the reduced form.

In vitro Translation of Trypanosoma cruzi Antigens Recognized by Human Chagasic Sera

Several reports indicate that infective (trypomastigotes) and noninfective (epimastigotes) forms of Trypanosoma cruzi display relatively simple antigenic patterns when analyzed by immunoprecipitation with hyperimmune sera from experimental animals. Thus, epimastigotes show, as their major antigen, an Mr 72,000 glycoprotein, whereas trypomastigotes (both blood forms and tissue culture-derived forms) display instead an Mr 90,000 surface glycoprotein (Nogueira et al., 1981, Journal of Experimental Medicine 153: 629-639; Snary et al., 1981, Molecular and Biochemical Parasitology 3: 343-356). These patterns become more complex when sera from chagasic patients are used in either immunoprecipitation techniques (Zingales et al., 1982, Molecular and Biochemical Parasitology 11: 111-124) or immunoblotting (Grogl and Kuhn, 1984, Journal of Parasitology 70: 822824). We have extended these studies and found that lysates of epimastigotes and tissue culturederived trypomastigotes subjected to immunoblotting with human chagasic sera, display reactive bands over a wide range of molecular weights (Mr 150,000 to Mr 12,000) (Rangel-Aldao et al., 1986, Molecular and Biochemical Parasitology 20: 25-32). Most of these antigenic bands were shared by both parasite forms and the differences in their antigenic patterns were only manifested upon antigen dilution. Trypomastigotes showed, predominantly, a group of bands in the range of Mr 150,000 to Mr 75,000, whereas epimastigotes displayed a cluster of bands in the range of Mr 72,000 to Mr 36,000 (Rangel-Aldao et al., 1986, loc. cit.). everal reports indicate that infective (trypoThis qualitative homology of epimastigotes and trypomastigotes could be exploited to produce in vitro, by the techniques of molecular biology, most of the common antigens of the infective trypomastigotes from the mRNA of epimastigotes that are not infectious and can be easily cultured in mass quantities. These antigens could then be used to develop sensitive tools for diagnosis and vaccination tests for Chagas' disease. As a prerequisite to achieve this goal, we have isolated polyadenylated (poly A+) RNA from epimastigotes, translated it in vitro in a wheat germ cell-free system, immunoblotted the translated products with a pool of sera from 10 patients with Chagas' disease, and showed that it is possible to translate in vitro antigens produced n vivo by epimastigotes. These antigens are also shared by tissue culture-derived trypomastigotes (Rangel-Aldao et al., 1986, loc. cit.). Poly A+ RNA was isolated from 1 x 1010 cultured epimastigotes (Rangel-Aldao et al., 1983, Journal of Biological Chemistry 258: 6979-6983), washed twice with 50 ml of 1% w/v NaCI, 0.6 t,g/ml cycloheximide, and 20% glycerol. The cells were resuspended in lysis buffer (50 mM TrisHC1, pH 7.4, 0.5% Triton X-100, 50% glycerol, and 1,000 units of human placental ribonuclease inhibitor) and lysed at -20 C with a Dounce homogenizer. The supernatant obtained from the lysis step was mixed with 3 volumes of a buffer solution containing 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) and the total cytoplasmic RNA extracted with phenol-chloroform (Perry et al., 1972, Biochimica et Biophysica Acta 262: qualitative hom logy of epimastigotes and astigotes could b exploited to produce itro, by the techniques of molecular biology, of the common antigens of the infective astigotes from the mRNA of epimastis that are not infectious and can be easily red in ma s quan ties. Th se antigens could e used to develop sensitive tools for disis and va cination te ts for Chagas' di ase. rerequisite to achieve this goal, we have

Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin.

Human placental ribonuclease inhibitor (PRI) abolishes both the ribonucleolytic activity of angiogenin toward 28S and 18S rRNA and its angiogenic activity on the chicken embryo chorioallantoic membrane. Treatment of the angiogenin-PRI complex with p-hydroxymercuribenzoate releases enzymatically active angiogenin. Assays measuring competition between angiogenin and bovine pancreatic ribonuclease A for PRI reveal that binding of the inhibitor to angiogenin is extremely tight, with a Ki value well below 0.1 nM. The stability of the angiogenin-PRI complex was assessed by cation-exchange HPLC quantitation of free angiogenin. No significant dissociation was detected after 17 hr at 25 degrees C in the presence of a large excess of bovine ribonuclease, which serves as a scavenger for free inhibitor. The results of these experiments, based on the predictive capacity of the angiogenin/RNase homology, suggest that PRI and related inhibitors may participate in the in vivo regulation of angiogenin and that this might have pharmacologic and/or therapeutic implications.

Specific transcription of preformed nucleoprotein complexes, containing the adenovirus major late promoter, with a chromatographic fraction containing RNA polymerase II.

Incubation in a HeLa whole-cell extract converted a plasmid DNA containing the adenovirus type 2 major late promoter into ordered nucleoprotein complexes similar to those described for simian virus DNA [Sinha, S. N., Hellwig, R. J., Allison, D. P. & Niyogi, S. K. (1982) Nucleic Acids Res. 10, 5533-5552]. Purified nucleoprotein complexes containing the plasmid DNA were able to serve as template for accurate transcription in vitro. Use of such nucleoprotein complexes eliminated the need for addition of nontemplate DNA (poly[d(I-C)]) to transcription reaction mixtures with template concentrations too low to yield a detectable specific-transcription signal from "naked" DNA. Of the four fractions resulting from phosphocellulose column chromatography of the whole cell extract, only the fraction that contained the RNA polymerase II activity was needed to accurately transcribe these nucleoprotein complexes in the presence of human placental ribonuclease inhibitor. In contrast, specific transcription of naked template DNA under these conditions required at least one additional fraction containing specificity factors. These results show that the nucleoprotein complexes contain factor(s) needed for specific initiation of transcription and that these complexes may be useful in the purification and analysis of these factors.

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli.

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.

Interaction of ribonuclease A with estrogen receptor from rat mammary tumor MTW9.

Incubation of crude estrogen receptor preparations from mammary tumor cytosol with RNase A increases the sedimentation coefficient of the receptor from 9.7 S to 10.4 S. The effect is not obtained with other low molecular weight basic proteins (lysozyme, cytochrome c, or histone H2B). Nonenzymically active RNase A derivatives such as performic acid oxidized RNase A, fully reductively methylated RNase A, carboxymethyl-His-119-RNase A, and RNase S-protein were ineffective. RNase T1, an acidic endoribonuclease, was also without effect. However, enzymically active RNase S', prepared from a mixture of RNase S-protein and S-peptide, shifted the sedimentation to 10.4 S. The increased sedimentation is not accompanied by a change in the Stokes radius of the receptor (74 A) or buoyant density in metrizamide (1.24 g/ml). The effect of RNase A on the sedimentation of the receptor can be reversed by subsequent incubation with human placental RNase inhibitor or with rabbit anti-RNase A antibodies. Direct interaction was shown by chromatography of the receptor on RNase A Sepharose. Thus, the shift in sedimentation results from binding of RNase A to the receptor and, although this requires that the enzyme active site be available, enzymic activity is not responsible for the effect. The interaction of RNase A with the receptor occurs at low ionic strength; it does not occur at elevated ionic strength or after activation of the receptor by precipitation with ammonium sulfate.

Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli.

An endoribonuclease which cleaves only single-stranded RNA has been purified from nucleoli of Ehrlich ascites tumor cells. The molecular weight of the ribonuclease is 50,000 to 52,000 as estimated from sedimentation in glycerol density gradients and by gel filtration on Sephadex G-100. The endoribonuclease requires Mg2+ or Mn2+ (0.2 mM) for optimum activity. Monovalent cations including K+, Na+, and NH+4 are inhibitory. The ribonuclease gave an apparent Km for single-stranded RNA of 30 microM. Using ribohomopolymers, we found that the enzyme could digest single-stranded, poly(C), poly(U), and poly(A) equally well, but would not degrade duplex poly(C) . poly(I) or poly(A) . poly(U). The lack of base specificity was further demonstrated using RNA sequence analysis of partial digest products of yeast 5.8 S RNA. The ribonuclease activity is sensitive to EDTA and N-ethylmaleimide, but is not inhibited by human placental RNase inhibitor. The enzyme makes endonucleolytic cleavages which generate 5'-phosphate-terminated oligonucleotides.

Identification of lysine residues in the binding domain of ribonuclease A for the RNase inhibitor from human placenta.

Amidination of the available lysine residues of the complex between RNase A and human placental RNase inhibitor has been performed with methyl acetimidate; the conditions of the derivatization preserve the complex functionally intact. Resistance of epsilon-acetimidyllysine residues to hydrolysis by trypsin allowed, after peptide mapping, the identification of lysine residues 7, 31, 41, 61, and 91 as those which were fully protected by the inhibitor from amidination. Lysine residue 37 was partially protected from amidination. In the presence of poly(A), lysine residues 41 and 61 of RNase A were fully protected from amidination, while lysine residues 7, 31, 37, 91, and 104 were only partially protected; the enzyme retained full activity. The results permit identification of lysine residues located in the binding domain of RNase A for the inhibitor. This region is not identical with, but does overlap, the binding domain for poly(A).

Effect of human placental ribonuclease inhibitor in cell-free ribosomal RNA synthesis

Exposure to purified placental ribonuclease inhibitor caused a marked reduction in ribonuclease activity associated with intact nucleoli. Addition of the placental RNase inhibitor to a nucleolar RNA synthesizing system, prior to the addition of ribonucleoside triphosphates, resulted in a significant increase in the size but did not alter the yield of the RNA products. The effect of the RNase inhibitor on the size of distribution of in, vitro transcriptional products could be reversed by the addition of N-ethylmaleimide after transcription was terminated. The properties of the ribonnclease(s) affected by the inhibitor were indicated by its resistance to N-ethylmaleimide and EDTA inhibition and by its limited endonucleolytic cleavage of the in, vitro transcript.

Role of mammalian RNase inhibitor in cell-free protein synthesis

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.