Structure–Function Study of Recombinant Onconase Variants

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in non-denaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure–function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.     

Effective expression and purification of recombinant onconase, an antitumor protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.     

Contribution of the C30/C75 disulfide bond to the biological properties of onconase

Onconase, a member of the pancreatic type ribonuclease family, is currently used as a chemotherapeutic agent for the treatment of different types of cancer. It is widely accepted that one of the properties that renders this enzyme cytotoxic is its ability to evade the cytosolic ribonuclease inhibitor (RI). In the present work, we produced and characterized an onconase variant that lacks the disulfide bond C30/C75. This variant mimics the stable unfolding intermediate des(30-75) produced in the reductive unfolding pathway of onconase. We found that the reduction of the C30/C75 disulfide bond does not significantly alter the cytotoxic properties of onconase, although the variant possesses a notably reduced conformational stability. Interestingly, both its catalytic activity and its ability to evade RI are comparable to wild-type onconase under mild reductive conditions in which the three disulfide containing intermediate des(30-75) is present. These results suggest that the C30/C75 disulfide bond could easily be reduced under physiological redox conditions.     

Thermal Stability of Onconase and Some Mutant Forms

Onconase is a small globular protein of the pancreatic ribonuclease superfamily. It is an important molecule because it possesses a selective antitumor activity. The interesting finding is that onconase has a high thermal stability, with a denaturation temperature close to 90d`C at p^H 6.0. A detailed comparison between the tertiary structures of onconase and bovine pancreatic ribonuclease has been accomplished in order to identify the molecular determinants of the high stability. The results of differential scanning calorimetry measurements confirm that the mutant forms of onconase, designed to be less stable than the parent enzyme, exhibit lower denaturation temperatures. In particular, the disulfide bridge at the C-terminus of onconase seems to play a pivotal role in stability.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Onconase: an unusually stable protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.     

Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase.

Onconase, a member of the ribonuclease superfamily, is a potent cytotoxic agent that is undergoing phase II/III human clinical trials as an antitumor drug. Native onconase from Rana pipiens and its amphibian homologs have an N-terminal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria, Onconase is isolated with an additional methionyl (Met1) residue and glutaminyl instead of a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated. The two reactions necessary for generating the pyroglutamyl residue have been monitored by MALDI-TOF MS. Results show that hydrolysis of Met(-1), catalyzed by Aeromonas aminopeptidase, is optimal at a concentration of >or= 3 m guanidinium-chloride, and at pH 8.0. The intramolecular cyclization of glutaminyl that renders the pyroglutamyl residue is not accelerated by increasing the concentration of denaturing agent or by strong acid or basic conditions. However, temperature clearly accelerates the formation of pyroglutamyl. Taken together, these results have allowed the characterization and optimization of the onconase activation process. This procedure may have more general applicability in optimizing the removal of undesirable N-terminal methionyl residues from recombinant proteins overexpressed in bacteria and providing them with biological and catalytic properties identical to those of the natural enzyme.     

Contribution of chain termini to the conformational stability and biological activity of onconase

Onconase, a member of the RNase A superfamily, is a potent antitumor agent which is undergoing phase III clinical trials as an antitumor drug. We have recently shown that onconase is an unusually stable protein. Furthermore, the protein is resistant to the action of proteases, which could influence its use as a drug, prolonging its biological life, and leading to its renal toxicity. Our investigation focused on the contribution of chain termini to onconase conformational stability and biological activities. We used differential scanning calorimetry, isothermal unfolding experiments, limited proteolysis, and catalytic and antitumor activity determinations to investigate the effect of the elimination of the two blocks at the chain termini, the N-terminal cyclized glutamine and the C-terminal disulfide bridge between the terminal Cys104 and Cys87. The determination of the thermodynamic parameters of the protein led to the conclusion that the two blocks at onconase chain termini are responsible for the unusual stability of the protein. Moreover, the reduced stability of the onconase mutants does not influence greatly their catalytic and antitumor activities. Thus, our data would suggest that an onconase-based drug, with a decreased toxicity, could be obtained through the use of less stable onconase variants.     

Expression and purification of recombinant human indoleamine 2, 3-dioxygenase

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression.     

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Expression of guinea-pig liver transglutaminase cDNA in Escherichia coli. Amino-terminal N alpha-acetyl group is not essential for catalytic function of transglutaminase

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. These enzymes are involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea-pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233-2, and produced the liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyl-terminal sequences. However, the alpha-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl alpha s1-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the N alpha-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.     

Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Abstract: A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli. The recombinant enzyme was purified to a specific activity of 500 μmol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely μ-helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved.     

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization

Onconase, an RNAse extracted from embryos of the Northern leopard frog ( Rana pipiens ), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an α-mating factor (α-MF)-prepro secretion signal, and an α-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L⁻¹ for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.     

Differential Requirement for the Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in RNA Damage-Induced Apoptosis in Primary and in Immortalized Fibroblasts

Onconase, an anticancer ribonuclease, damages cellular tRNA and causes caspase-dependent apoptosis in targeted cells (M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L. Newton, S. M. Rybak, and B. E. Magun. Cancer Res. 60, 1983–1994, 2000). The proapoptotic action of onconase depends on its RNase activity, but the molecular mechanisms leading to RNA damage-induced caspase activation are completely unknown. In this study, we have investigated whether onconase activates two signal-transduction pathways commonly stimulated by conventional chemo- and radiotherapy, namely the stress-activated protein kinase (SAPK) cascade and the pathway leading to the activation of nuclear factor-kappa B (NF-κB). We found that, in all cell types tested, onconase is a potent activator of SAPK1 (JNK1 and JNK2) and SAPK2 (p38 MAP kinase), but that it is incapable of activating NF-κB. Inhibition of p38 MAP kinase activity with a pharmacological inhibitor, SB203580, demonstrated that p38 MAP kinase is not required for onconase cytotoxicity. Using explanted fibroblasts from mice that contain targeted disruption of both jnk1 and jnk2 alleles, we found that JNKs are important mediators of onconase-induced cytotoxicity. Surprisingly, following the immortalization of these same cells with human papilloma virus (HPV16) gene products E6 and E7, additional proapoptotic pathways (exclusive of JNK) were provoked by onconase. Our results demonstrate that onconase may activate proapoptotic pathways in tumor cells that are not able to be accessed in normal cells. These results present the possibility that the cytotoxic activity of onconase in normal cells may be reduced by blocking the activity of JNKs.     

Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.     

Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus solfataricus in relation to starch processing

A gene (ssg) encoding a putative glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was cloned and expressed in Escherichia coli, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90 degrees C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated beta-d-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from S. solfataricus produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from S. solfataricus has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.     

Oxidative folding and N-terminal cyclization of onconase

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally.     

Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.

A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively. The enzyme binds riboflavin with a Kd of 1.2 microm. The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin. The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.