The Rana catesbeiana rcr Gene Encoding a Cytotoxic Ribonuclease

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5′- and 3′-RACE technique. The cDNA clone, named rcr gene, contained a 5′-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3′-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression inEscherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

Ribonucleases are widely found in living organisms and have been proposed to function in RNA metabolism and gene expression (1). Several abundant ribonucleases have been isolated from organs of various animals and have been well characterized. For example, various kinds of ribonuclease have been purified from bovine organs, e.g. pancreas, liver, kidney, brain, and seminal fluids. RNase A, from bovine pancreas, has been extensively characterized and is widely used in molecular biology (2). The occurrence of several homologous ribonucleases in different organs of the same animal suggests the existence of a family of homologous genes regulated in a tissue-specific fashion. Although these ribonucleases are abundant and well characterized biochemically, their biological significance is still not clear (3,4). Recently, several proteins with known biological functions were found to have intrinsic ribonucleolytic activity. For example, angiogenin from humans possesses both angiogenesis and ribonucleolytic activities (5). Eosinophil-derived neurotoxin and eosinophil cationic protein from humans exert both neurotoxicity and ribonucleolytic activity (6,7). Bovine seminal ribonuclease, a dimer made up of two identical 124 amino acid subunits, exerts both antitumor and ribonucleolytic activity (8). The ribonucleases from frog oocytes exert antitumor activity as well as ribonucleolytic activity, e.g. onconase from Rana pipiens (9 -11) and RC-RNase 1 from Rana catesbeiana (12,13).
Three distinct properties are found in the frog RNases. First, the substrate specificity of frog RNase is pyrimidine-guanine and that of mammalian RNase is pyrimidine-adenine (14). Second, RNase from the frog is resistant to RNase inhibitor from human placenta, whereas mammalian RNase is susceptible to the inhibitor. Third, the RNases of frogs with antitumor activity are found in oocytes, e.g. bullfrog RC-RNase (14), whereas most of mammalian RNases with cytotoxic activity are found in eosinophils. Cloning and site-directed mutagenesis of the ribonuclease gene and overexpression of the gene product will enable us to elucidate this difference more effectively than traditional chemical modification methods. However, prior to the present study, cloning of a frog RNase gene had not yet been successful, although some efforts were made. For example, a synthetic gene for onconase was made by oligonucleotide synthesis and assembly, and a semisynthetic gene for onconase was made by ligating a genomic DNA fragment with two synthetic DNA fragments. However, the recombinant products of this gene expressed in Escherichia coli was not enzymatically active (15,16). In this report, we present for the first time the successful cloning of ribonuclease cDNA from frog liver rather than from oocytes where RC-RNase is stored. We also describe the expression of active RC-RNase in E. coli and purification of recombinant RC-RNase to homogeneity. Site-directed mutagenesis of RC-RNase was made to determine the residues essential for catalytic activity and cytotoxicity.

EXPERIMENTAL PROCEDURES
Frogs-Native bullfrogs (R. catesbeiana) were obtained from a local frog farm and housed at 25°C. The frogs were 300 -400 g in body * This work was supported by Academia Sinica and the National Science Council of the Republic of China Grant NSC 85-2331-B-001-053. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF039104.
Bacterial Strains, Plasmids, and Oligonucleotides-E. coli BL21 (DE3) from Novagen (Madison, WI) was used for RC-RNase production. Plasmid pGEM-T from Promega (Madison, WI) was used for cDNA cloning by PCR method. Plasmid pET11d and pET22b from Novagen were used for construction of expression vector pET-pel-RC. Oligonucleotides synthesized by Perkin-Elmer were used as primers for PCR ( Table I).
Preparation of Total RNA-Total RNA from the liver of R. catesbeiana was isolated by the acid guanidinium thiocyanate/phenol/chloroform method (18). Briefly, 0.5 g of liver was frozen in liquid nitrogen and then ground by mortar. The powder was dissolved in guanidinium thiocyanate. After phenol/chloroform extraction, the RNA was precipitated with ice-cold isopropyl alcohol. The pellet was dissolved in 200 l of diethyl pyrocarbonate-treated water, and RNA concentration was determined by spectrophotometer at A 260 .
Purification of mRNA and cDNA Synthesis-Purification of mRNA (100 g in total) was performed using a Quickprep Micro mRNA Purification Kit from Pharmacia Biotech (Uppsala, Sweden). The synthesis of double-stranded cDNA was done using Marathon TM cDNA Amplification Kit from CLONTECH (Palo Alto, CA) according to the manufacturer's instruction manual.
PCR and cDNA Cloning of RC-RNase Gene-To clone the RC-RNase gene, degenerate primers for highly conserved regions of the amino acid sequence of the ribonuclease (RNase) family were designed to amplify a 220-base pair DNA fragment. The oligonucleotide RC-I, corresponding to residues 34 -41 (CKRVNTFI) of RC-RNase, and oligonucleotide RC-IIA, corresponding to residues 101-107 (PVHFAGI) of RC-RNase, were selected as primers. The selection of codon usage was based on the DNA sequence of the above-mentioned conserved peptides of bovine pancreatic RNase (19). The cycle protocol for PCR was as follows: 3 cycles of 94°C for 1 min, 42°C for 1 min, and 72°C for 1 min followed by 30 cycles of 94°C for 30 s, 45°C for 1 min, and 72°C for 50 s. A 220-base pair DNA fragment of PCR product was gel-purified and ligated with pGEM-T vector. The DNA sequence of the insert was analyzed, and its encoded amino acid sequence was identical to residues 34 -107 of RC-RNase from bullfrog oocyte. Later, two oligonucleotides (RC-5Ј-RACE and RC-3Ј-RACE) were selected as primers in combination with AP1 primer for 5Ј-RACE (rapid amplification of cDNA ends) and 3Ј-RACE to reach the 5Ј end and 3Ј end of the cDNA (CLONTECH). The PCR products of both 5Ј-RACE and 3Ј-RACE were gel-purified, ligated, cloned, and sequenced. The corresponding DNA fragments from 5Ј-RACE and 3Ј-RACE were fused by heat denaturation, annealing, and PCR amplification using AP1 adaptor as primer. Following fusion reaction between 3Ј-RACE and 5Ј-RACE products, the full-length cDNA fragment was ligated into pGEM-T vector.
Construction of Expression Vector-The strategy for expression of RC-RNase was to drive the T7-RNA polymerase promoter in E. coli B21(DE3) by isopropyl-␤-D-thiogalactopyranoside (IPTG) induction. For the construction of the expression vector, a pelB signal sequence was obtained from pET22b and inserted in front of the RC-RNase gene. The nucleotide sequence of the 3Ј end of the pelB sequence was GCG ATG GCC ATG G which encoded -Ala-Met-Ala2-Met-Xaa (Xaa may represent Val, Ala, Asp, Glu, and Gly), and the signal peptidase cleavage site located at the C terminus of the second Ala. If the gene was introduced directly into the vector through the NcoI site, two additional residues (Met-Xaa) would be added on the N terminus of the expressed protein. In this study, the NcoI site was moved 6 bases upstream (-GCG ATG GCC2ATG G 3 -GCC ATG GCT2NNN N; where N represents any nucleotide) by PCR method using a two-point mutated oligonucleotide (3Ј pelB) as primer without changing its encoded amino acids. The pelB gene and its upstream region were obtained by PCR method ranging from the XbaI site to the modified NcoI site. The cDNA of the mature protein region was amplified by PCR using oligonucleotides (5Ј Nco-MA, 3Ј Bam) containing the NcoI and BamHI sites, respectively, as primers. These PCR products containing the modified pelB gene and the RC-RNase gene were digested with NcoI, gel-eluted, and ligated by T4 DNA ligase. For amplification of the ligated DNA fragment, PCR was performed using oligonucleotides (5Ј Xba, 3Ј Bam) containing the XbaI and BamHI sites, respectively, as primers. The amplified pelB-RC-RNase ligated DNA fragment was introduced into the pET11d expression vector through the XbaI and BamHI sites and named pET-pel-RC.
Expression of RC-RNase in E. coli-The pET-pel-RC transformed cells were grown in TB medium containing 100 g/ml ampicillin to stationary phase (A 600 of 1.8) at 34 and 37°C before 0.5 mM IPTG induction. The cell pellets and culture media were collected after centrifugation. The cells were disrupted by sonication in buffer A (20 mM Hepes, pH 7.9, 0.5 mM EDTA). The supernatant and insoluble pellet of cell lysate were collected by centrifugation. The RC-RNase in supernatant, cell pellet, and culture medium was analyzed by Coomassie Blue staining, Western blotting, and zymogram assay.
Purification of RC-RNase-After 3 days' incubation the culture media (700 ml) were placed into dialysis bags and concentrated by polyethylene glycol (PEG) 20,000 absorption, followed by dialysis against buffer A (20 mM Hepes, pH 7.9, 0.5 mM EDTA). After dialysis, the crude samples were loaded onto a blue dextran Sepharose CL 6B column (1.5 ϫ 12 cm) and eluted by 160 ml of buffer A with 0 -0.4 M KCl gradient. Fractions containing RC-RNase were collected and dialyzed against buffer A and loaded on a buffer A-equilibrated carboxymethylcellulose column (1 ϫ 5 cm). The RC-RNase was eluted by 70 ml of buffer A with 0 -0.2 M KCl gradient. The protein components of each purification step were analyzed by SDS-PAGE followed by Coomassie Blue staining.
Mutagenesis of RC-RNase Gene-For single point mutation of RC-RNase at residues H10A and K35A, each individual mutated oligonucleotide 5Ј H10A and 5Ј K35A in combination with oligonucleotides 3Ј Bam were used as primers for amplification of a mega-fragment by PCR. The gel-purified mega-fragments and oligonucleotide 5Ј Nco-MA were further used as primers for amplification of the full-length mutated RC-RNase gene. With regard to mutation of RC-RNase at residues H103A, oligonucleotides 5Ј Nco-MA in combination with oligonucleotide 3Ј H103A were used as primers and performed similarly as described above for PCR. The PCR products were introduced into pET11d through the NcoI and BamHI sites.
DNA Sequence Analysis-DNA sequencing was performed by the dideoxy chain termination method using d-Rhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). The nucleotide sequence was analyzed by a Perkin-Elmer 377 automated DNA sequencer.
Northern Blotting Analysis-The mRNA transcripts coding for RC-RNase from each tissue and organ were determined by Northern blotting. Twenty g of total RNA from different organs or tissues of bullfrogs were fractionated by 1.2% agarose gel electrophoresis and blotted onto a Hybond-N ϩ nylon membrane from Amersham Corp. (Buckinghamshire, UK) in a mild alkaline transfer buffer (75 mM sodium citrate dihydrate, 10 mM NaOH, pH 11.5, 0.75 M sodium chloride) (20). After prehybridization, the blot was hybridized overnight with a biotin-labeled RC-RNase DNA fragment from NcoI-BamHI-digested pETpel-RC plasmid at 65°C in hybridization buffer (0.25 M sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS). The biotinylation of DNA probe was performed using a non-radioactive random octamer labeling system from Tropix (Bedford, MA). The signals on the membrane were detected by a Southern-Star TM Chemiluminescent Detection System according to the instruction manual provided by the manufacturer. Biotinylated RNAs from New England Biolabs (Beverly, MA) were used as RNA markers.
Protein Determination-Total protein of each purification step was determined by the Bradford method (21). The concentration of RC-RNase was estimated from Coomassie Blue-stained gels using bovine pancreatic RNase A as standard. The elution profile of each chromatography was detected by a UV monitor and recorder. The amino acid sequence of recombinant RC-RNase and its internal fragment were performed as described previously (12).
Ribonuclease Assay-The ribonuclease activity of each column eluate was determined by the ability for dinucleotide CpG cleavage, followed by thin layer chromatography (PEI-cellulose F) from Merck (Darms- tadt, Germany) and UV illumination at 254 nm (12). The ribonuclease activity of each purification step was determined by the release of acid-soluble nucleotides from bakers' yeast total RNA after ribonuclease digestion. One unit of enzyme activity is defined as the amount of enzyme producing one A 260 acid-soluble material at 37°C for 15 min (12). The ribonuclease activity from crude sample was analyzed by zymogram assay on RNA-casting PAGE (22). Briefly, after electrophoresis the gel was washed twice with 25% isopropyl alcohol in 10 mM Tris-HCl, pH 7.5, to allow SDS removal and protein renaturation. The activity was shown by incubating the gel at room temperature for 30 min in 10 mM Tris-HCl, pH 7.5, followed by 0.2% toluidine blue O staining. Gel Electrophoresis and Western Blotting-Proteins were separated by 13.3% SDS-PAGE and stained by Coomassie Brilliant Blue R (12). For Western blotting analysis, gel was contact-transferred to Hybond C nitrocellulose membrane (Amersham) in transfer buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 0.5 mM 2-mercaptoethanol) overnight. The membrane with immobilized proteins was blocked by 5% skim milk in phosphate-buffered saline and incubated with rabbit anti-RC-RNase antiserum (1:1,000 dilution) and goat anti-rabbit antibodies conjugated with alkaline phosphatase. RC-RNase was visualized by the addition of a bromochloroindolyl phosphate/nitro blue tetrazolium substrate (22).

Cloning of the Full-length of RC-RNase cDNA
Six clones with inserts of predicted size were obtained from the reverse transcription-PCR product of liver mRNA using oligonucleotides RC-I and RC-IIA as primers. The sequence of these six clones corresponded to amino acid residues 34 -107 of RC-RNase from frog oocytes. To obtain full-length cDNA, oligonucleotides RC-5Ј-RACE and RC-3Ј-RACE in combination with adaptor AP-1 were used as primers for PCR amplification of the 5Ј upstream and 3Ј downstream regions of the RC-RNase gene. There were eight clones obtained from 5Ј-RACE and six clones from 3Ј-RACE. After fusion of the 5Ј-RACE and 3Ј-RACE fragments, four clones with correct sequences in the pGEM-T vector were obtained and named pGEM-RC.
One cDNA clone, named rcr gene, contained an 883-nucleotide insert including a 31-base poly(A) tail. It had a single open reading frame with the first ATG initiation codon located at nucleotide 244 from the 5Ј end of the clone followed by a 399-nucleotide open reading frame and ending with an inframe termination codon TAG at position 642. The open reading frame encoded a 133-amino acid protein including a 22-amino acid putative signal peptide and a 111-amino acid RC-RNase (Fig. 1). The presumptive polyadenylation site, AATAAA, was located 121 nucleotides downstream from the TAG stop codon. The N-terminal residue was glutamine rather than pyroglutamate as found on RC-RNase. The encoded amino acid of the open reading frame was identical to that of RC-RNase from bullfrog oocytes except a 22-amino acid putative signal peptide was found at the N terminus (23).

Expression of RC-RNase mRNA in the Liver of Female Bullfrog
As shown in Fig. 2A, the RC-RNase mRNA was only expressed in the liver of female bullfrogs, and no signal was found in the liver of male bullfrogs. The RC-RNase mRNA was not found in other tissues or organs of female frogs, i.e. pancreas, kidney, stomach, intestine, ovary, and spleen. The ribosomal RNA content including 28 S rRNA and 18 S rRNA in each tissue and organ was similar except 28 S rRNA of oocyte was partially degraded as detected by ethidium bromide staining (Fig. 2B). This indicates that the rcr gene was specifically transcribed in the liver of female bullfrogs rather than in oocytes where it localized.

Expression and Purification of Recombinant RC-RNase
The RC-RNase gene with pelB signal sequence was subcloned in expression vector and named pET-pel-RC (Fig. 3). pET-pel-RC transformed E. coli BL21(DE3) cells were grown at 37 and 34°C to stationary phase (A 600 of 1.8). IPTG was added to a final concentration of 0.5 mM, and incubation was continued for another 24 h. Equal aliquots of total supernatant, insoluble pellet, and culture media were taken for analysis. The amount of total protein in culture media was quite low compared with that of the supernatant and insoluble pellet of cell lysate (Fig. 4A). Most of the mature RC-RNase protein was equally present in the supernatants and insoluble pellets, but the precursor of RC-RNase, which had slower mobility than RC-RNase on the gel, existed exclusively in the insoluble pellets upon IPTG induction (Fig. 4B). The insoluble pellets, which contained a large amount of RC-RNase, showed no activity, whereas the RC-RNase in the culture medium and supernatant, possibly derived from the periplasm of E. coli, was active (Fig. 4C). The ribonuclease activity in culture medium increased 5-10-fold when the temperature was shifted from 37 to 34°C. The activity did not increase further when the temperature dropped to 30°C. There was no apparent effect of IPTG induction on the production of secreted active RC-RNase in the culture medium. The production of secreted RC-RNase in the culture medium reached a plateau after 3 days' incubation as measured by both Western blotting and the acid-soluble method (data not shown).
The RC-RNase in the culture medium was concentrated by PEG absorption and purified by blue dextran-Sepharose CL6B and carboxymethylcellulose column chromatographies. RC-RNase protein seemed visible on the gel with PEG-concentrated crude samples, and it was homogeneous after being purified by two column chromatographies (Fig. 5A). The recombinant RC-RNase had similar antigenicity and identical mobil-ity as that of native RC-RNase from bullfrog as determined by Western blotting analysis (Fig. 5B). Both recombinant and native RC-RNase also showed similar specific activity and identical mobility as observed on the zymogram of RNA-casting SDS-PAGE (Fig. 5C). These results indicated that recombinant RC-RNase had similar properties to that of native RC-RNase. The results of purification after quantitation of ribonuclease activity at each purification step using the acid-soluble method are summarized in Table II. 2.55 mg of RC-RNase was purified from 120 ml of PEG-concentrated medium which came from the original 700 ml of culture medium. The yield of ribonuclease activity was 28.5% from the concentrated medium and 11% from the original culture medium. The specific activity of the recombinant RC-RNase was the same as that of native RC-RNase from bullfrog oocytes as determined by the acid-soluble method (12).

General Properties of Recombinant RC-RNase
Amino Acid Sequence Identity-The recombinant RC-RNase purified from E. coli culture media has a modified N terminus similar to that of native RC-RNase, and neither yield any signal upon Edman degradation. Therefore, the protein was fragmented with CNBr for internal sequence analysis. One peptide had a sequence of NVLST which was identical to residues 59 -63 of native RC-RNase and the corresponding amino acid sequence encoded by the cDNA.
Substrate Specificity-Only dinucleotides CpG and UpG were cleaved by the recombinant RC-RNase, whereas the others, i.e. UpA, ApU, CpC, UpU and CpA, were not cleaved under the same assay conditions. This property was identical to that of native RC-RNase.
Amino Acid Residues for the RNase Activity-Since residues His-12, Lys-41, and His-119 of RNase A are conserved in the RNase superfamily and are involved in catalysis of RNA degradation, the corresponding residues of RC-RNase, His-10, Lys-35, and His-103, were mutated to Ala. The recombinant proteins were purified to homogeneity by the same method as that of wild type RC-RNase (Fig. 6A). The antigenicity of mutated RC-RNase was the same as that of wild type RC-RNase (data not shown). The catalytic activities of recombinant H10A-RC-RNase and H103A-RC-RNase were completely lost, whereas the catalytic activity of recombinant K35A-RC-RNase was very low compared with that of wild type RC-RNase (Fig. 6B). These results indicate that RC-RNase and RNase superfamily genes possess similar catalytic residues.
Cytotoxicity-Both RNase activity and cytotoxicity exist in frog oocytic RNase. The cytotoxicities of native RC-RNase and recombinant RC-RNase against HeLa Tet-On cells were similar (Fig. 7). Nine percent of HeLa Tet-On cells survived when treated with native RC-RNase and 12% survived when treated with recombinant wild type RC-RNase at day 3 compared with untreated control cells. RNase activity-deficient H103A-RC-RNase was not toxic to the HeLa Tet-On cell. Typical phenomena of apoptosis were observed in the detached cells after RC-RNase treatment at day 2 and day 3, e.g. nuclear fragmentation, cytoplasmic blebbing, and DNA laddering. The surviving attached cells detached and floated into the medium after further RC-RNase incubation. 2 These results show that the residue His-103 is involved in both RNase activity and cytotoxicity.

Amino Acid Sequence and DNA Sequence Homologies in RNase Superfamily
The amino acid sequence of RC-RNase is homologous to those of frogs' RNases, i.e. 79.1% identity for Rana japonica oocytic RNase, 65.5% identity for R. catesbeiana liver RNase, and 52.4% identity for R. pipiens oocytic RNase (onconase) as calculated by the GCG-GAP program (15). It is not highly homologous to human eosinophil-derived neurotoxin (26.7% identity) and eosinophil cationic protein (25.0% identity), which are both cytotoxic to some neurons (6,7). The homologies of RC-RNase to other mammalian RNase genes are between 27.6 and 34.3% identity.
The DNA sequence homologies among frog RNase genes could not be compared because no DNA sequence data are available except for the RC-RNase gene. The homologies of RC-RNase to the RNase superfamily is between 41.5 and 47.9% when compared with 12 RNase genes by the GCG-GAP program. The relationship of these RNase genes is expressed as a phylogenetic tree created by the GCG-PILEUP program in Fig.  8. Amino acid sequence analysis indicated that RC-RNase is a cognate member in the frog RNase family but it is a distinct class in the RNase superfamily according to both amino acid sequence and DNA sequence. DISCUSSION The ribonuclease (RNase) gene superfamily combines functionally divergent proteins that share statistically significant  ). B, Western blotting analysis of RC-RNase. One-tenth aliquot of sample as described in A was taken for Western blotting analysis. The order of sample alignment was identical to that of A. C, ribonuclease activity assay on RNA-casting SDS-PAGE. One-fiftieth aliquot of sample as described in A was taken for activity assay. The order of sample alignment was identical to that of A.  (21) and that of step 3 sample was estimated on gel stained with Coomassie Brilliant Blue R using bovine pancreatic RNase A as standard.
b One enzyme unit is defined as the amount of enzyme producing one A 260 acid-soluble material under standard assay condition as described in the text. amino acid sequence similarity. Known members assigned to this family include secretory and nonsecretory RNases, angiogenin, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), antitumor protein (onconase), and RC-RNase from frog oocytes. Most of these genes in the superfamily have been cloned and sequenced and possess DNA sequence homology as shown in Fig. 8. The RNase gene from frogs has not previously been cloned and compared, although they possess several important properties, e.g. substrate specificity for pyrimidine-guanine and cytotoxic activity toward tumor cells. In this report, we succeeded in cloning RC-RNase gene and characterized the active residues for RNase activity and possible sites for cytotoxicity. From DNA sequence analysis, we found that RC-RNase is a distinct class in the RNase superfamily genes.
Onconase from R. pipiens oocytes combined with tamoxifen is being evaluated in human phase III clinical trials in patients with pancreatic carcinoma, and it is approaching maturity as a new member of the anticancer arsenal of drugs (16). However, a gene from R. pipiens has not been successfully cloned. A synthetic gene was made by oligonucleotide synthesis and assembly, and a semisynthetic gene was obtained by ligating a genomic DNA fragment with two synthetic DNA fragments. Those onconase genes expressed in E. coli and formed inclusion bodies. No RNase activity was observed until it was renatured by extensive steps. The specific activity of renatured onconase is 3-10% of native onconase from frog oocytes. In addition, an extra methionine is added on onconase's N terminus which is crucial for its RNase activity and cytotoxicity. The N-terminal methionine is removed by CNBr cleavage only if its internal methionine is substituted by other residues (15,16). In this report, a modified pelB signal peptide was introduced in front of RC-RNase; therefore, the expressed RC-RNase was secreted into the culture medium and was enzymatically active. There were still some RC-RNases with the same size as that of mature form RC-RNase that remained in soluble fractions and insoluble pellets of cell lysate, but they were not enzymatically active until denaturation and renaturation were performed. However, the renatured RC-RNase was not homogeneous because several minor bands with slower mobility than native RC-RNase were found on the RNA-casting gel (data not shown). Therefore, the establishment of a secretory expression system enabled us to obtain a recombinant RC-RNase comparable with native RC-RNase directly from culture media without further treatment.
Antitumor activity of RNase exists only in frog oocytic RNases and bovine seminal dimer RNase but not in other RNases, e.g. RNase A, bovine seminal monomer RNase, and bullfrog liver RNase. Neurotoxicity was found in human EDN, ECP, as well as in frog onconase. Chemical inactivation on RNase activity of onconase, EDN, and ECP by iodoacetate reduced the cytotoxicity/neurotoxicity of these proteins. These results indicate that RNase activity is essential to induce cytotoxic or neurotoxic action (9,11,24). However, two single base pair mutations (K35R, H128D) were introduced in human recombinant ECP to convert RNase inactivity and did not have discernible effect on antibacterial activity (25). This indicates that RNase activity is independent for antimicrobial activity of recombinant ECP. In the case of RC-RNase, our study showed that the wild type recombinant RC-RNase was cytotoxic to the HeLa Tet-On cells, and RNase activity-depleted H103A-RC-RNase was not cytotoxic to HeLa Tet-On cells. These results demonstrated that RNase activity is involved in cytotoxicity of RC-RNase against tumor cells. The amino acid residues of RC-RNase were modified by point mutation rather than chemical modification which might cause nonspecific modifications. Since RNase activity is involved in cytotoxicity of RC-RNase against tumor cells, specific receptors on tumor cell surfaces may recognize a receptor-binding domain on RC-RNase and allow for the entry of RC-RNase into the cytosol thus causing RNA degradation (26,27). The presence of specific receptors may explain the differential cytotoxicity of RC-RNase against tumor cells and normal cells. A receptor-binding domain may exist only in frog oocytic RNase, i.e. RC-RNase, that causes their cytotoxicity against tumor cells but does not exist in other HeLa Tet-On cells (1.5 ϫ 10 5 , CLONTECH) were seeded on 60-mm Nunc plates overnight in Dulbecco's modified Eagle's medium containing 10% fetal calf serum before 2 M RC-RNase treatments. Cell numbers were counted by trypan blue exclusion assay (13). q, cell without RC-RNase treatment; E, cells treated by native RC-RNase from frog oocytes; f, cells treated by recombinant wild type RC-RNase; Ⅺ, cells treated by H103A RC-RNase. Bars represent standard deviations of three experiments.
ribonucleases, e.g. RNase A. Successful cloning and series mutation of RC-RNase gene followed by RNase activity and cytotoxicity assay in a later study will enable us to find the receptor-binding domain of RC-RNase which may be crucial for understanding the cytotoxicity of RC-RNase.
Vitellogenin is a major yolk protein used for nutrition during embryogenesis. The vitellogenin gene is transcribed and translated in liver. The protein is secreted into the bloodstream and passed into the ovaries by receptor-mediated endocytosis and then stored in yolk granules of oocytes in X. laevis (28,29). In bullfrogs, the RC-RNase gene is transcribed in the liver, and the protein is closely associated with vitellogenin in the yolk granules of oocytes (22,30). No precursor form of RC-RNase is found in the oocyte, although a putative signal peptide exists in front of RC-RNase from DNA sequence analysis of the cloned gene. Therefore, it is suggested that RC-RNase is transcribed, translated, and processed in the liver, secreted into the bloodstream, passed into the ovaries by receptor-mediated endocytosis, and then stored in yolk granules. This is why the RC-RNase gene could be cloned from bullfrog liver rather than from oocytes. The possible function of RC-RNase in oocytes and early embryos may be involved in a defense mechanism against predators because of its cytotoxicity. The RNase activity and cytotoxicity to the oocyte itself is regulated by compartmentation in yolk granules and RNase-specific inhibitor binding in the cytosol to prevent self-attack of RNA and susceptible factors (22,30). Therefore, the absence of receptors on cell surfaces as well as the presence of inhibitors in the cytosol may also protect bullfrogs from self-attack in other tissues.