Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

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 Ki values of 2.9 x 10(-16) M and 6.8 x 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.     

Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity.

Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions, 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

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.     

Binding of placental ribonuclease inhibitor to the active site of angiogenin

The importance of specific residues in angiogenin for binding to placental ribonuclease inhibitor (PRI) has been assessed by examining the interaction of angiogenin derivatives with PRI. PRI binds native angiogenin with a Ki value of 7.1 X 10(-16) M [Lee, F. S., Shapiro, R., & Vallee, B. L. (1989) Biochemistry 28, 225-230]. Substitution of a Gln for Lys-40 in angiogenin by site-specific mutagenesis decreases the association rate constant 3-fold and increases the dissociation rate constant 440-fold, resulting in a 1300-fold weaker Ki value. The half-life of the mutant.PRI complex is 3.4 h compared to approximately 60 days for the native angiogenin.PRI complex. The magnitude of the change in Ki value suggests that in the complex, Lys-40 forms a salt bridge or hydrogen bond with an anionic moiety in PRI. Carboxymethylation of His-13 or His-114 with bromoacetate increases the Ki value 15-fold, and oxidation of Trp-89 by means of dimethyl sulfoxide and hydrochloric acid increases it 2.4-fold, suggesting that these residues also form part of the contact region with PRI. The changes in Ki value reflect an increase in the dissociation rate constant. On the other hand, dinitrophenylation of either Lys-50 or Lys-60 with 1-fluoro-2,4-dinitrobenzene does not significantly alter the Ki value, suggesting that these residues are not part of the contact region. These results indicate that PRI inhibition minimally involves the three residues critical for the activity of angiogenin--Lys-40, His-13, and His-114--and to a lesser extent its single tryptophan, Trp-89.     

Isolation of bovine angiogenin using a placental ribonuclease inhibitor binding assay

Angiogenin, which induces the formation of new blood vessels, was isolated previously from two human sources--HT-29 tumor conditioned media and normal plasma. By use of a newly developed binding assay, a similar protein has now been purified from bovine plasma at levels of 30-80 micrograms/L. This protein has the structural, enzymatic, and biological characteristics expected for an angiogenin molecule. Its amino acid composition is similar to that of the human protein, and 22 of 31 residues in the amino-terminal sequences are identical, including a block of 11 consecutive residues. Like human angiogenin, the bovine protein binds placental ribonuclease inhibitor, is inactive toward conventional RNase A substrates, and displays selective ribonucleolytic activity toward some rRNAs. In addition, the bovine protein induces angiogenesis in vivo in the chick embryo chorioallantoic membrane assay at levels as low as 44 fmol per egg. Thus, angiogenin is present in bovine sera at levels similar to those observed in man, and its enzymatic and biological activities are identical with those of the human protein.     

An in vitro binding assay for angiogenin using placental ribonuclease inhibitor

A convenient in vitro assay for angiogenin has been developed which greatly facilitates its routine detection and quantitation. The assay is based on the capacity of angiogenin to bind placental ribonuclease inhibitor (PRI); it is less tedious and more versatile than existing procedures that measure blood vessel growth or cleavage of rRNA. The test sample is added to a reaction mixture containing a known quantity of PRI, which complexes any angiogenin present in the sample. A slight excess of RNase A, relative to PRI, is then added, and the amount of RNase A which remains unbound is determined by measuring the generation of acid-soluble fragments from yeast RNA. The assay is sensitive to 30 fmol of angiogenin and is linear over a 17-fold concentration range. Use of the binding assay in parallel with a conventional RNase A assay provides a means of detecting angiogenin in chromatographic fractions and differentiating it from RNases. This procedure makes possible the isolation of angiogenin from new sources, such as nonhuman sera. It may also be applicable to other biologically active proteins with sequence homology to RNase A, e.g., eosinophil cationic protein or eosinophil derived neurotoxin.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.     

Primary structure of an alkaline ribonuclease from bovine liver

A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).     

Primary structure of a non-secretory ribonuclease from bovine kidney

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).     

Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats

Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of onconase with the human ribonuclease inhibitor protein

One of the tightest known protein-protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease inhibitor protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with K_i = 0.15 μM in a solution of low salt concentration.     

Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations

Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell‐specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long‐term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang‐based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang‐based hCFPs. Here we perform in total 2.7 µs replica‐exchange molecular dynamics simulations to investigate some of these mutations—G85R/G86R (GGRR_mut), Q117G (QG_mut), and G85R/G86R/Q117G (GGRR/QG_mut). GGRR_mut turns out to perturb greatly the overall Ang‐RNH1 interactions, whereas QG_mut optimizes them. Combining QG_mut with GGRR_mut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRR_mut‐based hCFPs resist RNH1 inhibition remarkably, Ang WT‐ and Ang QG_mut‐based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QG_mut‐based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.     

Primary structure of a base non-specific ribonuclease from Rhizopus niveus

The primary structure of a base non-specific ribonuclease from Rhizopus niveus (RNase Rh) was determined by nucleotide sequence analysis of the DNA fragment encoding RNase Rh gene including signal peptide sequence, and amino acid sequence analysis of the peptide obtained from RNase Rh and RNase Rh' (a protease-modified RNase Rh created during the course of purification). The sequence determined was: MKAVLALATLIGSTLASSCSSTA LSCSNSANSDTCCSPEYGLVVLNMQWAPGYGPANAFTLHGLWPDKCSGAYAPSGGCDSN RASSSIASVIKSKDSSLYNSMLTYWPSNQGNNNVFWSHEWSKHGTCVSTYDPDCYDNYE EGEDIVDYFQKAMDLRSQYNVYKAFSSNGITPGGTYTATEMQSAIESYFGAKAKIDCSSG TLSDVALYFYVRGRDTYVITDALSTGSCSGDVEYPTK (the sequence of signal peptide is underlined). The sequence indicates that the homology with the sequence of RNase T2 from A. oryzae with the same base specificity is about 42% and that the sequences around the two histidine residues which are supposed to be involved in the active site are fairly conserved.