title: Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties.
abstract: Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
authors.affiliation: Intramural Research Support Program, SAIC Frederick, Macromolecular Structure Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Newton
authors.forename: L
authors.initials: L
authors.lastname: Boque
authors.forename: A
authors.initials: A
authors.lastname: Wlodawer
authors.forename: C Y
authors.initials: CY
authors.lastname: Huang
authors.forename: S M
authors.initials: SM
authors.lastname: Rybak
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002474:Cell-Free System / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004527:Egg Proteins / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects; D015202:Protein Engineering; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D012156:Reticulocytes; D012260:Ribonucleases / Q000235:genetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D004527:Egg Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D012343:RNA, Transfer; D012260:Ribonucleases; C058703:ranpirnase