SEARCH

SEARCH BY CITATION

Keywords:

  • catalytic activity;
  • cysteine residues;
  • disulfide bond;
  • RNase κ;
  • site-directed mutagenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.


Abbreviations
DTT

dithiothreitol

IAA

iodoacetemide

NEM

N-ethylmaleimide

β-ΜΕ

β-mercaptoethanol

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

RNA metabolism is a determining factor for the maintenance of cell homeostasis. Dynamic functional roles have been attributed to RNA molecules, which are separate from encoding or participation in protein synthesis. Numerous studies have shown that non-protein-coding RNAs possess crucial roles for gene expression control, as well as regulation of the cell’s complex programmed responses to stimuli [1]. Since the fate of RNA molecules can be significantly affected by ribonucleases, the study of this highly diverse enzyme group is becoming increasingly important to better understand basic cellular processes such as proliferation and apoptosis or even their alterations leading to pathological conditions such as cancer.

Current RNase research has unveiled an increasing number of as yet unknown enzymes involved in a variety of biological pathways associated with cytoplasmic and nuclear RNA degradation, RNA maturation, RNA interference, antiviral defense etc. [2,3]. Novel or well-characterized RNases, apart from their ribonucleolytic activities, have been found to possess fundamental and diverse biological roles, such as angiogenic, neurotoxic, antitumor and immunosuppressive activities [4–6]. It is noted that a large number of ribonucleolytic enzymes with yet unidentified biological functions are constantly being discovered.

Extensive studies performed by our group have recently led to the establishment of a new family of ribonucleases, designated as the RNase κ family [7]. This family is widely represented in the animal kingdom, as counterparts are distributed from the phylum of Cnidaria to the class of mammals. All protein family members are highly conserved and consist of 95–101 amino acids, while the mammalian representatives are almost identical to each other. In all available metazoan genomes only one copy of the RNase κ gene is detected, consisting of two introns and three exons in almost all cases [8]. In the insect Ceratitis capitata, two RNase κ mRNA isoforms, deriving from alternative polyadenylation, were identified in all tissues and developmental stages. Sequence analysis of the extended 3′ UTR of the longer transcript revealed the existence of destabilizing elements, generating an attractive hypothesis that alternative polyadenylation could be regulating RNase κ expression in C. capitata [8].

The gene of the human counterpart of the RNase κ family is expressed as one main transcript in almost every human tissue and developmental stage as well as in a great number of carcinomas, an observation that implies a significant biological function for this enzyme. The human RNase κ is an endoribonuclease consisting of 98 amino acids which mainly hydrolyzes ApG and ApU phosphodiester bonds, as determined using synthetic radiolabeled RNA substrates [7].

It is well established that cysteine residues in ribonucleases may have distinct roles. In many RNases, such as RNase A, RNase T1 and RNase T2 family members [9,10], as well as in the bacterial RNases Sa, Sa2 and Sa3 [11], cysteine residues participate in the formation of disulfide bonds, which are necessary for their structural stability or activity. On the other hand, the requirement for disulfide bonds is not universal among RNases [12], while in some cases cysteine residues are involved in the catalytic activity [13]. In the present study, we investigated the importance of cysteine residues for the activity of human RNase κ, in order to gain insight into the structure and function of this enzyme.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Effect of reducing and alkylating agents on human RNase κ enzyme activity

Alignment of the amino acid sequences of RNase κ from different animal species expanding from sea anemone to humans revealed that all protein homologs contain 3–5 cysteine residues, three of which are completely conserved [8]. Conserved cysteines usually participate in the formation of intramolecular and intermolecular disulfide bonds, but they can also fulfill a wide range of different functions including metal binding, electron donation, hydrolysis and redox catalysis [14,15]. In order to investigate the role of cysteine residues in human RNase κ, we examined the effect of the reducing agent dithiothreitol (DTT), as well as the alkylating agents N-ethylmaleimide (NEM) and iodoacetamide (IAA), on the enzyme activity. The ribonucleolytic activity of human RNase κ was determined by incubation of the purified recombinant protein with a 5′-32P-labeled synthetic 30-mer RNA, whose sequence is cited in Experimental procedures, followed by denaturing gel electrophoresis and autoradiography. As shown in Fig. 1 (lanes 4–8), the reducing agent DTT in concentrations >2 mm causes complete inactivation of the enzyme, implying that the formation of at least one disulfide bond is absolutely necessary for its activity. On the other hand, NEM treatment (lanes 12–14) or IAA treatment (lanes 15–17) showed no effect on the enzyme activity or base specificity, even at the highest dose used (100 mm). The results obtained strongly suggest that free thiol groups are not implicated in the catalytic activity of human RNase κ.

image

Figure 1.  Effect of alkylating and reducing agents on the catalytic activity and base specificity of the human RNase κ. The 5′-labeled oligoribonucleotide 5′-CCCCGAUUUUAGCUAUCUGGGUUCAACUUG-3′ was incubated without RNase κ (lanes 1 and 9), in alkaline hydrolysis buffer (lanes 2 and 10) or with 1 U of ribonuclease T1 (lanes 3 and 11). The same amount of radiolabeled probe was incubated with 150 pg of the recombinant enzyme in 20 mm potassium phosphate, pH 7.0, containing 5 mm EDTA at 37 °C for 30 min (lane 4); or in the presence of 1, 2, 5, 20 mm DTT (lanes 5–8 respectively); 20, 50 and 100 mm NEM (lanes 12–14 respectively); and 20, 50 and 100 mm IAA (lanes 15–17 respectively). The reaction products were separated by 8 m urea 17% PAGE and visualized by autoradiography.

Download figure to PowerPoint

Human RNase κ contains intramolecular disulfide bond(s)

A number of ribonucleases have been shown to contain intramolecular disulfide bonds, while the bovine seminal ribonuclease (BS-RNase) is the only known RNase which forms homodimers that are linked through intermolecular disulfide bonds [16]. The human RNase κ was therefore analyzed by SDS/PAGE under both reducing and non-reducing conditions. Proteins containing intramolecular disulfide bonds are known to migrate faster than totally reduced proteins, due to the more compact nature of their structure [17–19]. The purified recombinant protein gave only a single band with an apparent molecular mass of approximately 14 kDa under reducing conditions (Fig. 2, lane 4). In the absence of β-mercaptoethanol (β-ME), the unreduced protein migrated more rapidly than the fully reduced form (Fig. 2, lane 1). When purified protein samples were treated with 5% or 10%β-ΜΕ, both forms of human RNase κ appeared (Fig. 2, lanes 2 and 3 respectively). Since no oligomers were detected in the totally unreduced sample, the above data strongly suggest that human RNase κ does not form homopolymers linked through disulfide bonds and contains at least one intramolecular disulfide bond.

image

Figure 2.  Effect of β-ME on the migration of the human RNase κ in SDS/urea PAGE. Equal amounts of the purified recombinant protein were treated with SDS loading buffer containing 6 m urea in the absence of β-ME (lane 1), or in the presence of 5% v/v (lane 2), 10% v/v (lane 3) and 20% v/v (lane 4) β-ME, at 37 °C for 30 min and then heated at 65 °C for 10 min. Samples were analyzed in a 17.5% SDS/polyacrylamide gel containing 6 m urea and the protein bands were visualized by silver staining. A set of marker proteins with known molecular weight were treated with 6 m urea and run in parallel (lane M).

Download figure to PowerPoint

Expression and purification of Cys-replacement variants in yeast

Human RNase κ contains five cysteine residues, of which Cys6, Cys14 and Cys69 are absolutely conserved among the family orthologs. We investigated the role of Cys residues in disulfide bond formation and in catalytic activity by constructing mutants in which the codons corresponding to cysteine residues in the plasmid were individually substituted with serine codons by site-directed mutagenesis of the pPICZaA/RNase κ cDNA (paA) construct. The serine substituted variants were expressed in Pichia pastoris and maximal production of the recombinant proteins occurred 2 days after methanol induction. In all experiments, the wild-type RNase κ and Cys variants were expressed at comparable levels and the produced recombinant proteins were purified as described in Experimental procedures. Following SDS/PAGE, a single sharp band exhibiting the expected molecular mass was detected by silver staining in all cases (Fig. 3A). The identity of the purified recombinant proteins was immunologically verified using a specific polyclonal antibody against an oligopeptide of the human RNase κ (Fig. 3B). No immunodetection was obtained in a parallel control experiment, where only the secondary antibody was used (data not shown).

image

Figure 3.  SDS/PAGE and western blot analysis of the wild-type and cysteine variants of human RNase κ. Equal amounts of the purified wild-type and mutant C6S, C7S, C14S, C69S and C85S recombinant proteins were analyzed by SDS/PAGE followed by silver staining (A) or by immunoblotting (B). A set of marker proteins of known molecular weights were run in parallel (lane M).

Download figure to PowerPoint

Cysteine residues that are involved in the formation of an intramolecular disulfide bond

Aiming towards the investigation of the number and connectivity of disulfide bond(s) in human RNase κ, we examined the effect of serine substituted variants in disulfide bond formation by reducing/non-reducing SDS/PAGE analysis. As shown in Fig. 4, under non-reducing conditions the fully oxidized C7S, C14S and C85S mutant recombinant proteins migrated faster than their totally reduced forms, demonstrating that these variants maintain intramolecular disulfide bonding. In contrast, C6S and C69S mutant recombinant proteins showed no mobility variation under reducing and non-reducing conditions, confirming that Cys6 and Cys69 form an intramolecular disulfide bond, a finding that is also supported by the observation that these cysteine residues are strictly conserved among the family members [8].

image

Figure 4.  Effect of β-ME on the migration of the human RNase κ cysteine variants in SDS/urea PAGE. Equal amounts of the purified recombinant variants C6S, C7S, C14S, C69S and C85S were treated with SDS loading buffer containing 6 m urea in the absence of β-ME (−) or in the presence of 20% v/v β-ME (+), at 37 °C for 30 min and then heated at 65 °C for 10 min. Samples were analyzed in a 17.5% SDS/polyacrylamide gel containing 6 m urea and the protein bands were visualized by silver staining.

Download figure to PowerPoint

Characterization of Cys-replacement variants for catalytic activity

We previously reported that the recombinant human RNase κ preferentially hydrolyzes the ApG phosphodiester bonds, while the ApU, UpA and UpU bonds are cleaved at a lower rate [7]. To examine the effect of the Cys mutations on the catalytic activity of RNase κ, we initially analyzed the activities of the wild-type and its variants using the 5′-CCCCGAUUUUAGCUAUCUGGGUUCAACUUG-3′, 5′-32P-labeled synthetic RNA as substrate. Three of the five Cys variants, C7S, C14S and C85S, were found to retain the catalytic activity and base specificity of the wild-type enzyme, as shown by the cleavage pattern demonstrated in Fig. 5 (lanes 4, 6, 7 and 9). On the other hand, the C6S and C69S mutations resulted in complete loss of enzymatic activity (Fig. 5, lanes 5 and 8).

image

Figure 5.  Ribonucleolytic activity and base specificity of wild-type and cysteine variants of human RNase κ. Equal amounts of the purified RNase κ (lane 4) and the recombinant variants C6S, C7S, C14S, C69S and C85S (lanes 5–9 respectively) were incubated with the 30-mer 5′- radiolabeled oligoribonucleotide 5′-CCCCGAUUUUAGCUAUCUGGGUUCAACUUG-3′ at 37 °C for 30 min and the reaction products were analyzed as described in Experimental procedures. Reactions performed in the absence of RNase κ, in alkaline hydrolysis buffer or in the presence of RNase T1 were also analyzed in parallel (lanes 1–3 respectively).

Download figure to PowerPoint

In order to verify the above data, we used the octanucleotide 5′-dTdTdTrAdGdTdTdT-3′ as a more specific substrate for the determination of the enzymatic activity of the wild-type and the Cys variants in relation to enzyme concentration and incubation time. This oligonucleotide contains only the preferred human RNase κ cleavage site (ApG) and therefore precludes any non-specific substrate hydrolysis that could be caused by possible contamination with pancreatic type RNases in the reaction mixture. C7S, C14S and C85S variants hydrolyzed the initial substrate almost completely after 60 min of incubation with the same overall rate of hydrolysis as in the case of the wild-type enzyme (Fig. 6A). No cleavage was detected in the case of C6S and C69S variants even after 60 min of substrate incubation with a five-fold higher amount of recombinant protein (Fig. 6B).

image

Figure 6.  Ribonucleolytic activity of the wild-type and cysteine RNase κ variants in relation to incubation time and enzyme concentration. The 5′-labeled 8-mer 5′-dTdTdTrAdGdTdTdT-3′ was incubated at 37 °C with 1 ng of the purified wild-type or mutant C6S, C7S, C14S, C69S and C85S recombinant protein for 0, 10, 20, 30 and 60 min (A) or with 0, 1.25, 2.5, 3.75 and 5 ng of each recombinant enzyme for 60 min (B). The reaction products were then analyzed as described in Experimental procedures.

Download figure to PowerPoint

Taken together, the above results led to the conclusion that Cys7, Cys14 and Cys85 are not essential for the activity of human RNase κ, even though Cys14 is completely conserved in all organisms examined. Moreover, our study demonstrates that the Cys6-Cys69 disulfide bond is critically important to the folding of human RNase κ into its functional conformation.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Ribonucleases are ubiquitous enzymes, broadly distributed in almost all organisms. They are classified in several families on the basis of their structural, catalytic and biological properties. Previous work from our laboratory has led to the establishment of a novel family of ribonucleases, designated as the RNase κ family, which is represented in a wide range of metazoans, expanding from Cnidaria to humans [8,20]. The extreme amino acid conservation between RNase κ orthologs in combination with their wide distribution imply a significant, as yet unknown, biological role for the RNase κ family enzymes. The molecular cloning and biochemical characterization of the RNase κ family human representative have also been recently described [7]. In the present work we report the role of cysteine residues on the function and disulfide bond formation of the human enzyme.

Among the conserved residues in the RNase κ orthologs are 3–5 cysteine residues. Amino acid sequence alignment indicates that Cys6, Cys14 and Cys69 (numbering according to the human representative) are absolutely conserved in all species examined. The other two cysteine residues (Cys7, Cys85) seem to have become established later in chordate evolution and are completely conserved only among the vertebrate classes of amphibians, birds and mammals [8].

As a first step in exploring the role of cysteine residues, we showed that the modification of free sulfhydryl groups of the human RNase κ via alkylation has no effect on enzyme activity. This finding does not constitute an unexpected observation, since no RNases have as yet been reported to rely on reduced cysteines for the catalysis of RNA hydrolytic reactions. However, the Escherichia coli RNase Τ has been found to be extremely sensitive to sulfhydryl reagents, suggesting that cysteine residues may be implicated in enzyme activity [21]. Later work has shown that two of the four cysteine residues are actually important for RNase T activity but do not directly participate in RNA catalysis. Specifically, the first residue (Cys168) has a strong influence on the molecule’s stability and activity [13,22], by virtue of its hydrophobic properties, while Cys112 has less of an effect on RNase T activity, but in this case the nucleophilic properties of the –SH group appear to be of paramount importance [13].

Proceeding with the further investigation of the role of cysteine residues in human RNase κ, experimental data from SDS/PAGE analysis under reducing and non-reducing conditions revealed that the recombinant protein appears as a monomer, migrating faster than the totally reduced form, indicating the intramolecular nature of any disulfide bonds in this enzyme. The loss of activity even at low concentrations of reducing reagents is in agreement with the thesis that one or more disulfide bonds is required to stabilize the enzyme in an active conformation.

The role of cysteine residues in disulfide bond formation and in catalytic activity was additionally examined by constructing variants in which the cysteine residues were individually replaced by serine. We were able to produce equal levels of the wild-type human RNase κ and Cys mutant forms in the P. pastoris expression system. When variants were tested for activity, our results demonstrated that two Cys residues are critical for the maintenance of an active enzyme conformation. Namely, the substitution of Cys6 and Cys69 by serines leads to complete inactivation of the human enzyme, while variants C7S, C14S and C85S are ribonucleolytically active. Furthermore, the fact that replacement of Cys7, Cys14 and Cys85 with serine had no effect on base specificity and on the kinetics of human RNase κ suggested that these residues may not be directly involved in substrate binding, although Cys14 is completely conserved in all representatives. The above results in combination with the migration pattern of the mutant recombinant proteins on SDS/PAGE, under reducing and non-reducing conditions, constitute strong evidence that Cys6 and Cys69 form an intramolecular disulfide bond, which is required for the folding of human RNase κ into its functional conformation. Given the fact that these cysteine residues are absolutely conserved in all RNase κ family members, it is conceivable that an intramolecular disulfide bond is not unique to the human representative but rather is conserved among the family orthologs.

Although the requirement for disulfide bonds is not universal among ribonucleases [12], disulfide bonds may be required for protein stability or primarily contribute to RNase activity [23,24]. More specifically, bovine RNase A contains four interweaving disulfide bonds, which are conserved in all 40 of the known sequences of homologous mammalian pancreatic RNases [25]. It has been shown that the two terminal disulfide bonds, restricting the N and C termini, are most important for conformational stability. On the other hand, the two embedded disulfide bonds, that are proximal to the active site, contribute to the proper alignment of the catalytic residues and are therefore necessary for efficient catalysis [26]. Both of the conserved disulfide bonds in the RNase T1 family enzymes (Cys2-Cys10 and Cys6-Cys103) are required for its conformational stability, but are only moderately important for enzyme activity [27]. It is interesting that an RNase T1 derivative, with both disulfide bonds broken, retains 35% of the activity of the native protein, while the enzymatic activity remains almost unchanged when the Cys2-Cys10 disulfide bond is disrupted [28]. The small bacterial RNases Sa, Sa2 and Sa3 contain a single disulfide bond linking cysteine residues near the ends of the molecules, e.g. residues 7 and 96 in RNase Sa [29]. When this disulfide bond is disrupted, although the proteins remain folded their Tm values are significantly lowered [11]. Finally, all the members of the RNase T2 family also include two conserved disulfide bonds, which are possibly required for their conformational stability and/or activity [30]. Two additional disulfide bonds are present only in the animal/plant RNase T2 subfamily, while the RNase LE from tomato is the only family member with five disulfide bonds [31].

Since no data concerning the structural conformation or the thermodynamics of folding of human RNase κ are yet available, no conclusion can be drawn about whether the Cys6-Cys69 disulfide bond is directly required for enzyme activity. An indication that this disulfide bond may be involved in the correct protein folding stems from the observation that during our efforts to express the recombinant enzyme in bacteria the formation of insoluble aggregates was observed [7]. The bacterial cytoplasm is normally even more reducing than its eukaryotic counterpart and consequently is not a good environment for the production of disulfide bonded proteins, which tend to misfold and aggregate forming inclusion bodies [32].

The formation of disulfide bonds in proteins requires a sufficiently oxidizing environment [33]. At the present time, it is clear that complex enzymatic systems catalyze disulfide bond formation in proteins in the endoplasmic reticulum, in mitochondria or in the bacterial periplasm [34]. Disulfide bonds are therefore common to extracellular proteins, or to proteins inserted into membranes [35]. This observation is in agreement with the existence of disulfide bonds in the RNase T1 and RNase A families, in the bacterial Sa ribonucleases, as well as in some of the RNase T2 family members which are typically secreted from the cell. Disulfide bonded RNases from the T2 family can also be found in membrane-bound compartments which are more oxidizing than the cytosol, such as the lysosome or the vacuole [36].

The finding that human RNase κ comprises an intramolecular disulfide bond, in combination with the fact that among the family orthologs the conserved residues are mainly clustered in two hydrophobic domains at the C and N termini, may suggest a possible membrane localization for this molecule, but this in no case constitutes evidence for the cellular topology of the enzyme. One of the first transmembrane proteins that have been found to exhibit ribonucleolytic activity is the Ire1p, a type I endoplasmic reticulum transmembrane protein that plays a key role in the unfolded protein response signaling pathway in eukaryotic cells and exhibits two distinct catalytic activities, a kinase activity and an endoribonucleolytic activity [37].

In the present study, we demonstrate the critical role of distinguished cysteine residues on the enzyme activity of human RNase κ through the formation of an intramolecular disulfide bond. Additional investigations of the enzyme protein structure by X-ray crystallography may further support the findings presented here and add insight into the catalytic mechanism of human RNase κ.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Materials

Oligonucleotide synthesis and sequence analysis were performed by VBC-Genomics (Vienna, Austria). Restriction enzymes and T4 polynucleotide kinase were purchased from New England Biolabs (Hitchin, UK). Plasmid preparation kits and Protino His-Bind Resin were from Macherey-Nagel (Düren, Germany). GS115 P. pastoris host cells and the yeast expression vector pPICZaA were purchased from Invitrogen (Carlsbad, CA, USA). The QuikChange™ Site-Directed Mutagenesis Kit was from Stratagene (Santa Clara, Canada). [γ-32P] ATP was obtained from Izotop Ltd (Obninsk, Russia). RNase T1, Q-Sepharose Fast Flow and the alkaline phosphatase conjugated goat anti-chicken IgG were purchased from Sigma-Aldrich (St Louis, MO, USA). Marker proteins for SDS/PAGE molecular weight estimation were obtained from Fermentas Life Sciences (Vilnius, Lithuania). All other reagents used were of analytical grade and were purchased from Merck (Darmstadt, Germany).

Preparation of human RNase κ variants

Wild-type human RNase κ cDNA was cloned into the yeast expression vector pPICZαΑ, constructing the paA plasmid, as previously described [7]. Mutations replacing cysteine by serine (C6S, C7S, C14S, C69S and C85S) were created by primer-directed mutagenesis using the QuikChange™ Site-Directed Mutagenesis Kit, according to the manufacturer’s recommendations. The following oligonucleotides were used as sense primers (mutations underlined) to amplify the human RNase κ from paA, using standard PCR conditions with Pfu DNA polymerase: C6S, 5′-GCG TCG CTC CTG ΑGC TGT GGG CCG AAG-3′; C7S, 5′-CG CTC CTG TGC TCT GGG CCG AAG CTG-3′; C14S, 5′-G AAG CTG GCC GCC AGC GGC ATC GT -3′; C69S, 5′-G CAA GTC AGC TAC AAC TCT TTC ATC GCT GCA GGC-3′; C85S, 5′-GGA GGC TTC TCT TTC AGC CAA GTT CGG CTC-3′. Oligonucleotides with complementary sequence were used as antisense primers. All constructs were sequenced in both directions to verify the introduction of the desired mutations, using the 5′AOX or 3′AOX universal primers. Multiple clones were sequenced to compensate for misreading.

The wild-type and mutant constructs were integrated into the genome of P. pastoris GS115 host cells by electroporation as described by the manufacturer’s manual, and the screening for cDNA integration was conducted by PCR analysis of yeast chromosomal DNA as previously described [7]. Selection of the transformed clones, producing efficient amounts of recombinant proteins, was performed as follows. A number of randomly selected recombinant colonies was inoculated in 5 mL buffered complex glycerol medium (1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 4 × 10−5% biotin, 1% glycerol) and incubated at 30 °C in a shaking incubator (250 r.p.m.) until the culture reached D600 = 2–6 (approximately 16–18 h). The cells were harvested by centrifuging at 1500 g for 5 min at room temperature and re-suspended, in order to induce expression, in an appropriate volume of buffered complex methanol medium (1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 4 × 10−5% biotin, 0.5% methanol) so that D600 was 1–1.2. The cultures were maintained for 3 days and supplemented daily with 0.5% v/v methanol. One milliliter of each culture was harvested every day and the collected supernatants were clarified by centrifugation (10 min, 10 000 g, 4 °C) and stored at –80 °C until tested. The presence of the recombinant RNase κ in the supernatants was detected by dot-blot analysis using a polyclonal antibody against the human protein [7]. Large-scale production of wild-type and mutant recombinant proteins was performed under similar conditions using the colonies that rendered the best yield in the small-scale experiments.

Purification of the wild-type human RNase κ and mutant recombinant proteins

Wild-type RNase κ and the cysteine variants were purified from 2-day culture supernatant. Then 50 mL of extracellular medium were exhaustively dialyzed against buffer A (20 mm Tris/HCl, pH 8.0, 1%Νonidet P-40) and loaded onto a Q-Sepharose Fast Flow column (1 mL) pre-equilibrated with the same buffer. After washing the column with buffer A, the RNase was eluted with 15 mL of a linear gradient 0–0.25 m NaCl in the same buffer. One milliliter fractions were collected and the fractions containing the larger amount of recombinant protein, as estimated by dot-blot analysis, were pooled. The pooled fractions were diluted 1 : 4 with IBW buffer (50 mm sodium phosphate–NaOH, pH 8.0, 300 mm NaCl) and loaded onto a Protino His-bind Resin column (0.5 mL) pre-equilibrated with IBW buffer. After washing with IBW buffer, the recombinant protein was eluted with the same buffer containing 250 mm imidazole and dialyzed against 20 mm potassium phosphate buffer (pH 7.0). The concentration of the purified proteins was estimated according to Bradford [38], using bovine serum albumin as standard.

SDS/PAGE and western blot analysis

The wild-type and mutant recombinant proteins were analyzed by SDS/PAGE according to Laemmli [39] and protein bands were visualized by silver staining. 17.5% SDS polyacrylamide gels containing 6 m urea were used to analyze the migration pattern of the purified proteins under reducing and non-reducing conditions. For immunodetection, proteins were blotted onto a nitrocellulose membrane [40], which was subsequently incubated with a specific polyclonal antibody raised in chicken as mentioned previously [7]. Primary antibody–antigen complexes were detected using goat anti-chicken IgG conjugated to alkaline phosphatase and developed by 5-bromo-4-chloro-indolyl phosphate/nitroblue tetrazolium.

Ribonucleolytic activity and base specificity assay

The determination of ribonucleolytic activity or base cleavage sites of the wild-type enzyme and cysteine variants C6S, C7S, C14S, C69S and C85S was performed by incubation of the purified proteins with 5′-[γ-32P] ATP-labeled synthetic 8-mer 5′-dTdTdTrAdGdTdTdT-3′ or 30-mer 5′-rCrCrCrCrGrArUrUrUrUrArGrCrUrArUrCrUrGrGrGrUrUrCrArArCrUrUrG-3′ in 20 mm potassium phosphate buffer (pH 7.0) containing 5 mm EDTA at 37 °C for the appropriate time. The one nucleotide ladder was prepared by alkaline hydrolysis of the 30-mer 5′-end-labeled substrate in a buffer containing 0.5 mm EDTA, 75 mm sodium carbonate buffer (pH 9.2) at 90 °C for 20 min. RNase T1 digestions were conducted by incubating the 30-mer probe in 15 mm sodium citrate buffer (pH 5.0), 1 mm EDTA and 3.5 m urea with 1 U of RNase T1 at 55 °C for 15 min. All reactions were stopped with an equal volume of loading buffer containing 5 mm Tris, 5 mm boric acid, 0.05% bromophenol blue and 10 m urea. The reaction products were analyzed on 17% denaturing polyacrylamide gel containing 8 m urea and visualized by autoradiography.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work is part of the PhD thesis of Ms Kiritsi and was supported by a Faculty Research Grant from the National Kapodistrian University of Athens.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References