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Glycosylation of onconase increases its conformational stability and toxicity for cancer cells

Byung-Moon Kim1, Hana Kim, Ronald T Raines

  • 1Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.

Biochemical and Biophysical Research Communications
|February 27, 2004
PubMed

Insights

Glycosylated Onconase (gONC) protein, produced using Pichia pastoris, shows increased cancer cell toxicity. This enhanced stability and efficacy suggest potential for improved cancer chemotherapy.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Onconase (ONC) is a cancer chemotherapeutic agent in Phase III trials.
  • The non-glycosylated form of ONC is the only one identified, despite a known N-linked glycosylation site.

Purpose of the Study:

  • To produce recombinant glycosylated ONC (gONC) using the Pichia pastoris expression system.
  • To investigate the structural and functional differences between glycosylated and non-glycosylated ONC.

Main Methods:

  • Utilized Pichia pastoris for recombinant protein expression.
  • Analyzed protein structure using Circular Dichroism (CD) spectroscopy.
  • Assessed protein stability via melting temperature (Tm) and proteinase K resistance.

Main Results:

  • Successfully produced approximately 10 mg/L of ONC, with 80% glycosylated at N69.
  • CD spectroscopy confirmed identical secondary structures for ONC and gONC.
  • gONC exhibited a high-mannose core structure.
  • Glycosylation significantly increased ONC's toxicity to K-562 cancer cells (50-fold lower IC50).
  • gONC demonstrated increased Tm and proteinase K resistance compared to ONC.

Conclusions:

  • Recombinant glycosylation of ONC at N69 enhances its cytotoxic activity against cancer cells.
  • The increased conformational stability of gONC likely contributes to its elevated efficacy.
  • This study provides a foundation for developing more potent glycosylated ONC-based cancer therapies.

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