SINGLE AMINO-ACID SUBSTITUTIONS AT THE N-TERMINUS OF A RECOMBINANT CYTOTOXIC RIBONUCLEASE MARKEDLY INFLUENCE BIOCHEMICAL AND BIOLOGICAL PROPERTIES

Onconase is a cytotoxic ribonuclease with antitumor properties. A semi synthetic 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 a cid 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-termin al Met amino acid residue. In this study, we demonstrate that modifica tion of the 5'-region of the gene to encode [Met(-1)]Ser or [Met-(-1)] Tyr instead of the native pyroglutamate results in recombinant onconas e derivatives with restored activities. [Met-(-1)]rOnc(E1Y) was more a ctive than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with t he action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibit or of protein synthesis in the cell-free rabbit reticulocyte lysate as say, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinan t onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (oncona se is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1) ]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma c ells with IC50's very similar to that of onconase (IC50 3.5, 10, and 1 0 mu g/mL after 1 day and 0.16, 0.35, and 2.5 mu g/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 d erivatives was potentiated by monensin, N(H)4Cl, and retinoic acid. Br efeldin 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-con taining medium in the presence or absence of cells at 37 degrees C sho wed that the recombinant proteins were as stable to temperature and ce ll culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amin o acid residue containing a hydroxyl group produces recombinant protei ns with ribonuclease and cytotoxic properties similar to native oncona se.