Single Amino Acid Substitutions at the N-Terminus of a Recombinant Cytotoxic Ribonuclease Markedly Influence Biochemical and Biological Properties
详细信息 查看全文
- 作者：Dianne L. Newton ; Lluis Boque ; Alexander Wlodawer ; Charles Y. Huang ; and Susanna M. Rybak
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 14, 1998
- 年：1998
- 卷：37
- 期：15
- 页码：5173 - 5183
- 全文大小：139K
- 年卷期：v.37,no.15(April 14, 1998)
- ISSN：1520-4995
文摘
Onconase is a cytotoxic ribonuclease with antitumorproperties. A semisynthetic gene encodingthe entire protein sequence was constructed by fusing oligonucleotidescoding for the first 15 and the last6 of the 104 amino acids to a genomic clone that encoded the remainingamino acid residues [Newton,D. L., et al. (1997) Protein Eng. 10, 463-470].The resulting protein product expressed inEscherichiacoli exhibited little enzymatic or cytotoxic activity due to theunprocessed N-terminal Met amino acidresidue. In this study, we demonstrate that modification of the5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the nativepyroglutamate results in recombinant onconase derivativeswith restored activities. [Met-(-1)]rOnc(E1S) wasmore active than [Met-(-1)]rOnc(E1Y) in allassaystested. Consistent with the action of native onconase,[Met-(-1)]rOnc(E1S) was a potent inhibitorofprotein synthesis in the cell-free rabbit reticulocyte lysate assay,degrading tRNA at concentrations thatcorrelated with inhibition of protein synthesis. An interestingdifference between the recombinant onconasederivatives and the native protein was their susceptibility toinhibition by the major intracellular RNaseinhibitor, PRI (onconase is refractory to PRI inhibition).[Met-(-1)]rOnc(E1S) and[Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cellswith IC50's very similar to that ofonconase (IC50 3.5, 10, and 10 
g/mL after 1 day and0.16, 0.35, and 2.5 g/mL after 5 days for onconase,[Met-(-1)]rOnc(E1S), and[Met-(-1)]rOnc(E1Y), respectively). Similar to thatof onconase, cytotoxicactivity of the recombinant derivatives was potentiated by monensin,NH4Cl, and retinoic acid. BrefeldinA completely blocked the enhancement of cytotoxicity caused by retinoicacid with all three proteins.Thus, drug-induced alterations of the intracellular trafficking ofthe recombinant derivatives also resemblesthat of onconase. Stability studies as assessed inserum-containing medium in the presence or absence ofcells at 37 
C showed that the recombinant proteins were as stable totemperature and cell culture conditionsas the native protein. Therefore, exchanging the Glu amino acidresidue at the amino terminus of onconasewith an amino acid residue containing a hydroxyl group producesrecombinant proteins with ribonucleaseand cytotoxic properties similar to native onconase.
g/mL after 1 day and0.16, 0.35, and 2.5 g/mL after 5 days for onconase,[Met-(-1)]rOnc(E1S), and[Met-(-1)]rOnc(E1Y), respectively). Similar to thatof onconase, cytotoxicactivity of the recombinant derivatives was potentiated by monensin,NH4Cl, and retinoic acid. BrefeldinA completely blocked the enhancement of cytotoxicity caused by retinoicacid with all three proteins.Thus, drug-induced alterations of the intracellular trafficking ofthe recombinant derivatives also resemblesthat of onconase. Stability studies as assessed inserum-containing medium in the presence or absence ofcells at 37 C showed that the recombinant proteins were as stable totemperature and cell culture conditionsas the native protein. Therefore, exchanging the Glu amino acidresidue at the amino terminus of onconasewith an amino acid residue containing a hydroxyl group producesrecombinant proteins with ribonucleaseand cytotoxic properties similar to native onconase.