Bovine Ribonuclease Research Articles

N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.

Identification of a Fungal Protein ofSyncephalastrum racemosumas Aspartic Proteinase

During purification of fungal deoxyribonuclease (DNase) fromSyncephalastrum racemosum,a protein which was functionally unknown and persistently existed in the DNase-containing fractions through chromatography over DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose was identified. The protein was finally separated from DNase after affinity chromatography on a cibacron blue-Sepharose column and purified to apparent homogeneity after gel chromatography on a Superdex 200 HR column. Ten tryptic peptides of this protein were isolated and sequenced. Searching in the sequence data bank with the aid of the computer program PC/Gene, we found that this protein was highly homologous to aspartic proteinases, such as pepsin and rhizopuspepsin. Because of its fungal origin and because the protein indeed showed catalytic cleavage on peptide bonds of bovine serum albumin, RNase, and carbonic anhydrase, we termed this protein syncephapepsin. The molecular weight of syncephapepsin is 38,000 daltons, based on gel filtration and sodium dodecyl sulfate–polyacrylamide electrophoresis.

The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates.

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.

Multimode High-Performance Liquid Chromatography of Fluorescently Labeled Oligosaccharides from Glycoproteins

A polymeric, secondary amine stationary phase was used to develop a method for the separation of 2-aminobenzamide (AB)-labeled neutral and anionic oligosaccharides from glycoproteins. Sequential hydrophilic interaction liquid and anion exchange chromatography were performed in the same chromatographic analysis (multimode HPLC) and unambiguous separation between neutral and anionic oligosaccharides was accomplished. Improved resolution was also achieved. The oligomannosidic-type structures from bovine ribonuclease B (Man4GlcNAc-AB–Man9GlcNAc-AB) were separated not only by size, but also by the branch location of terminal Manα(1 → 2)-linked residues. Sialylated lactosamine-type oligosaccharides from human α1acid glycoprotein were resolved according to charge, Fuc content, and the number of Galβ(1 → 4)Galβ(1 → 3) repeats. The minor fucosylated and polylactosamine species were well separated from the major sialylated tetra-antennary oligosaccharides. Volatile mobile phases were used to minimize sample handling for matrix assisted laser desorption mass spectrometric analysis of peak fractions. Novel polylactosamine structures (3–5 repeats) from α1-acid glycoprotein were deduced from the molecular weight analysis. Multimode HPLC should prove useful for preparing low pmol quantities of fluorescently labeled oligosaccharides with fewer steps and minimal sample handling for mass spectrometric analysis, important requisites for structural studies of sample-limited glycoproteins.

A Procedure for the Large-Scale Isolation of Major Bovine Seminal Plasma Proteins

A method for the stepwise purification of bovine seminal plasma proteins aSFP, PDC-109, TIMP-2, RNAse, and BSP-30K/PDC-109 complex is presented. It is based on the use of nondenaturing, classical chromatographic procedures. The isolation protocol is reproducible, inexpensive, easy to carry out in a common laboratory, and yields large amounts of pure proteins for biochemical and structural studies.

Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.

Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase.

The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary forms, MxM and M=M, of the enzyme reported earlier [Piccoli, R., et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1870-1874]. We found that the inhibitor does not interact with the intact isolated forms but has dramatic, differential effects on the two forms when the assays are performed under reducing conditions. These conditions, which are essential for full activity of the inhibitor, and are typical of its cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD). The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive to PRI. The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein. An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein with a shift in the equilibrium between the two forms towards the dimer. These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment housing the PRI and the ribosomal RNA, the molecular target of the enzyme.

Residual structure in unfolded proteins revealed by Raman optical activity.

Because of its ability to probe directly the chiral elements of the peptide backbone, together with the very short time scale of the scattering process, vibrational Raman optical activity (ROA) can provide new information on structure in non-native states of proteins. Here we report ROA studies of hen egg white lysozyme and bovine ribonuclease A in unfolded denatured states, prepared by reducing all the disulfide bonds. ROA spectra of unfolded lysozyme at 45, 20, and 2 degrees C, and of unfolded ribonuclease A at 35 and 20 degrees C, are presented and discussed. At 45 and 20 degrees C, unfolded lysozyme appears to contain very little extended secondary structure, but at 2 degrees C there could be roughly 20% of the native amount of alpha-helix present but little beta-sheet. Unfolded ribonuclease A, on the other hand, appears to contain roughly 50% of its native-like secondary structure, including both alpha-helix and beta-sheet, at 20 degrees C; similar secondary structure persists at 35 degrees C, but the amount is reduced. The most striking result is the observation of three sharp ROA bands in the extended amide III region, originating in coupled C alpha-H and N-H deformations, which might monitor directly the dominant intrinsic propensities for residues to adopt particular phi, psi angles, averaged over the different amino acids in the mobile heteropolypeptide. Specifically, positive bands at approximately 1300 and 1314 cm-1 appear to monitor propensities for alpha-helix and beta-structure, respectively, and a negative band at approximately 1237 cm-1 appears to monitor that for the poly(L-proline) II helix. These signals are generated by individual residues clustering in the most favorable regions of the Ramachandran plot and are present even in the absence of signals from the corresponding extended secondary structures. At 45 degrees C, the 1300 and 1314 cm-1 ROA bands of unfolded lysozyme coalesce into a single sharp band from which an analysis similar to that used for exchange effects in NMR suggests a rate of approximately 2.6 x 10(12) s-1 for interconversion between the individual residue conformations at this temperature.

The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 Å resolution

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.

Conformational Effects on Reversed-Phase Chromatography of Proteins with Particle Beam LC/FT-IR Spectrometry and Free Solution Capillary Electrophoresis

In this study, reversed-phase HPLC (RPC), particle beam FT-IR spectrometry, and capillary electrophoresis (CE) were employed to ascertain the dynamic conformational structure empirically of various forms of bovine ribonuclease A (RNase A) to investigate the interdependence of protein conformation and RPC separation. RNase A was analyzed in four conformational states: native, partially denatured, completely unfolded, and completely unfolded and disulfide-reduced (denatured). Each form was analyzed individually and produced similar multizoned RPC profiles composed of several low-response peaks before the primary peak. Particle beam FT-IR spectrometry results from the RPC fractions of the partially denatured and unfolded forms were identical to one another and exhibited a loss of ordered structure. The infrared spectra of the RNase A that was introduced into the HPLC in the denatured and reduced form showed an excess of β-sheet content, particularly the primary peak. We believe this to be a non-native form of RNase A. CE migration times of RNase A samples that were reduced in 2-mercaptoethanol and unfolded in increasing concentrations of urea increased with the degree of unfolding. Mobilities of RNase A RPC fractions were compared to those of the urea unfolded samples. The migration time of the primary RPC fraction of the denatured form showed an intermediate migration rate between that of the native, spherical geometry and denatured, open geometry forms. It is unclear whether this β-structure formed during column propagation or upon elution.

P 181 Immunosuppressive effect of bovine spermal ribonuclease in animal keratoplasty models

P 181 Immunosuppressive effect of bovine spermal ribonuclease in animal keratoplasty models

Immunosuppressive effect of bovine spermal ribonuclease in animal keratoplasty models.

Immunosuppressive effect of bovine spermal ribonuclease in animal keratoplasty models.

Structure of the crystalline complex of deoxycytidylyl-3',5'-guanosine (3',5'-dCpdG) cocrystallized with ribonuclease at 1.9 A resolution.

The X-ray structure of bovine ribonuclease A cocrystallized with the dinucleotide deoxycytidylyl-3',5'-guanosine has been determined at 1.9 A resolution and refined by restrained least squares to R = 0.218 for 7807 reflections. The structure established that the recently observed retrobound mode of attachment of substrate analogues cytidylyl-2',5'-guanosine and deoxycytidylyl-3',5'-guanosine found in soaked RNase A crystals is also present in the cocrystallized complex. Retrobinding is thus unlikely to be the result of restrictions imposed by the crystalline environment as the ligands soak into the lattice but rather a phenomenon specific to small nucleotides containing guanine.

Full Antitumor Action of Recombinant Seminal Ribonuclease Depends on the Removal of Its N-Terminal Methionine

Bovine seminal RNase (BS-RNase) is a dimeric member of the pancreatic-like ribonuclease superfamily, with antitumor activity. We report here that recombinant Met(-1) BS-RNase is a less potent cytotoxic factor, while structurally and catalytically indistinguishable from BS-RNase isolated from natural sources. Mature recombinant BS-RNase instead displays full antitumor action. This suggests that the conformation of the N-terminal region of BS-RNase is among the structural determinants of its antitumor action, in addition to its catalytic activity and its quaternary structure.

A Study of the Intracellular Routing of Cytotoxic Ribonucleases

Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.

Anti-HIV activity of bovine seminal RNase

Anti-HIV activity of bovine seminal RNase

Molecular characterization of surface topology in protein tertiary structures by amino-acylation and mass spectrometric peptide mapping.

Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins.

Transglycosylation Activity of Mucor Hiemalis Endo-β-N-Acetylglucosaminidase Which Transfers Complex Oligosaccharides to the N-Acetylglucosamine Moieties of Peptides

A novel endo-β-N-acetylglucosaminidase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-N-acetylglucosaminidase, Endo-M, could liberate the complex type of asparagine-linked oligosaccharides by hydrolysis of diacetylchitobiose linkage from glycoproteins. The treatment of Endo-M with N-acetylglucosamine and asialotransferrin glycopeptide having the complex type of oligosaccharides resulted in the transfer of the released oligosaccharide from the glycopeptide to N-acetylglucosamine. The structure of the product after transfer was deduced to be (GlcNAc)2-Man-(Gal-GlcNAc-Man)2 by a combination method of pyridylamination and high perfomance liquid chromatography, and mass-spectrometry. The enzyme could transfer the complex type of oligosaccharide from asialotransferrin glycopeptide to peptidyl-N-acetylglucosamine prepared from bovine ribonuclease with the high-mannose type of oligosaccharide. This will lead to the construction of neoglycoproteins containing different types of oligosaccharides.

Complex between Bovine Ribonuclease A and Porcine Ribonuclease Inhibitor Crystallizes in a Similar Unit Cell as Free Ribonuclease Inhibitor

We obtained three different morphologies of crystals of bovine ribonuclease A and porcine ribonuclease inhibitor. X-ray quality crystals were grown in 1·3 M ammonium sulfate, 100 mM sodium acetate (pH 5·0) and 20 mM dithiothreitol at 21°C. These crystals have the symmetry of the tetragonal space group I4 with a=133·3 Å and c =86·7 Å and diffract to 2·5 Å resolution; they have the same symmetry and only slightly different cell parameters than the crystals of free ribonuclease inhibitor. Polyacrylamide gel electrophoresis and the crystal density indicate that both ribonuclease inhibitor and ribonuclease A are present in the crystals. Although small, crystals are suitable for three-dimensional structural analysis.

Human Pancreatic-Type and Nonpancreatic-Type Ribonucleases: A Direct Side-by-Side Comparison of Their Catalytic Properties

The catalytic properties and substrate preference of several highly purified human ribonucleases from different organs and body fluids have been examined in detail using various low-molecular-weight compounds and single- or double-stranded polyribonucleotides as substrates. All single-stranded polyribonucleotides were degraded by nonpancreatic-type (npt) RNases at a slower rate than by pancreatic-type (pt) enzymes: ptRNases were 20 times more active on RNA and poly(U) substrates and more than 6000 times more active on poly(C). Pancreatic-type RNases degraded poly(C) faster than RNA, showing a strong preference for poly(C) over poly(U) with the following activity ratios: RNA/poly(C), 0.44; RNA/poly(U), 12; poly(C)/poly(U), 27, In contrast, nptRNases cleaved RNA more rapidly than synthetic homopolymers, preferring poly(U) over poly(C) with the following ratios: RNA/poly(C), 130; RNA/poly(U), 10; poly(C)/poly(U), 0.08. Human ptRNases degraded poly(A) and double-stranded polyribonucleotides about 100 and 400 times faster, respectively, than bovine RNase A. However, no measurable activity could be detected on these substrates with nptRNases. The activities of ptRNases on dinucleoside phosphates (CpN and UpN) or uridine and cytidine 2′,3′-cyclic phosphates were similar to those of bovine RNase A; nptRNases, instead, cleaved only CpA and UpA at an appreciable rate. The effects of pH, ionic strength, and divalent cations on the activity of these ribonucleases were also investigated using yeast RNA as a substrate.