Glycosylation of onconase increases its conformational stability and toxicity for cancer cells.
Onconase (ONC) is currently in Phase III clinical trials as a cancer chemotherapeutic agent. Despite the finding that ONC contains an N-linked glycosylation site (-N69-V70-T71-), only the non-glycosylated form of the protein has been identified to date. We employed the Pichia pastoris expression system to produce recombinant glycosylated ONC (gONC) protein. Approximately 10 mg of ONC protein was secreted per liter of culture media, of which about 80% was glycosylated at N69. CD spectroscopic analyses revealed that the secondary structure of gONC is identical to that of ONC. We found that gONC contains a high-mannose core structure. Importantly, glycosylation of ONC at N69 greatly increased its toxicity for K-562 cancer cells. Specifically, the IC50 value of gONC was 50-fold lower than that of ONC. Glycosylation increased both the Tm of ONC and its resistance to proteinase K, suggesting that the elevated cytotoxicity of gONC is related to higher conformational stability.
Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.
Byung-Moon
BM
Kim
Hana
H
Kim
Ronald T
RT
Raines
Younghoon
Y
Lee
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D019286:Endopeptidase K / Q000378:metabolism; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000378:metabolism / Q000494:pharmacology; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013696:Temperature
D000970:Antineoplastic Agents; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011994:Recombinant Proteins; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D019286:Endopeptidase K; C058703:ranpirnase