Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Abstract

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

Figure 1

Identification and purification scheme of multiple ribonucleases in bullfrog oocytes. (A) SDS–PAGE analysis of phosphocellulose column eluates. The ribonucleases in the yolk granules of oocytes were extracted by 0.09 M NaCl and subjected to fractionation by column chromatography on phosphocellulose and equal aliquots were taken for SDS–PAGE and Coomassie blue staining. (B) Purification scheme of ribonucleases from bullfrog oocytes. RC, RC 2, RC 3, RC 4, RC 5 and RC 6 represent RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, respectively. P-11, phosphocellulose (Whatman); CM52, carboxymethyl cellulose (Whatman); Heparin, heparin Sepharose 4B (Pharmacia); Blue, blue dextran Sepharose 4B (Pharmacia); Mono S, FPLC mono S (Pharmacia).

Figure 2

Analyses of multiple purified ribonucleases from bullfrog. (A) SDS–PAGE analysis. Purified ribonucleases (2 µg each) were separated by 13.3% SDS–PAGE and stained by Coomassie blue. (B) Western blotting. Purified ribonucleases (0.1 µg each) were separated by 13.3% SDS–PAGE, transferred to nitrocellulose membranes and probed with anti RC-RNase antibody. (C) Zymogram analysis. Ribonucleases (5 ng each) were separated by RNA-casting 13.3% non-reducing SDS–PAGE and stained by toluidine blue O. Lane A, bovine pancreatic ribonuclease A; lane L1, bullfrog liver-specific ribonuclease (RC-RNase L1); lanes RC, RC 2, RC 3, RC 4, RC 5 and RC 6 represent RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, respectively.

Figure 3

Base specificity of purified bullfrog ribonucleases toward a synthetic RNA substrate. A 5′-end-labeled oligoribonucleotide was partially digested with ribonucleases as indicated in Figure 2 at 30°C for 10 min, separated by 15% urea–PAGE and visualized by autoradiography. C, 5′-³²P-labeled RNA substrate; OH^–, RNA substrate treated with 0.05 M sodium bicarbonate-carbonate, pH 9.2, at 90°C for 4 min. The RNA sequence and cleavage sites are shown in the left and right margins, respectively.

Figure 4

Phylogenetic tree of ribonuclease genes. The DNA sequences of the following ribonuclease genes were clustered under the pileup and figure commands in the GCG program: bullfrog oocyte ribonucleases (RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 6), onconase from R.pipiens (32), human eosinophil-derived neurotoxin (34), bovine pancreatic RNase (35), human pancreatic RNase (36) and human angiogenin (37).

Figure 5

Amino acid sequence alignment of ribonucleases from Rana spp. frogs. RJ-lectin, the lectin or ribonuclease from oocytes of R.japonica (12); onconase, the ribonuclease from the oocytes of R.pipiens (12). The amino acid sequences were analyzed and aligned under the commands of pileup and prettybox in the GCG programs. Asterisks indicate conserved amino acid residues for catalytic activities of ribonuclease superfamily.

Figure 6

Concentration-dependent cytotoxicities of bullfrog ribonucleases toward HeLa S-3 cells. Cells (3 × 10³) were seeded on a 96-well plate overnight and incubated with bullfrog ribonucleases and bovine pancreatic ribonuclease for 48 h (six wells per treatment). The survival rates of ribonuclease-treated HeLa S-3 cells were counted by ATP-Lite M (Packard BioScience Company) and expressed as a percentage.