Glycosylation of onconase increases its conformational stability and toxicity for cancer cells Byung-Moon Kim, a,1 Hana Kim, a Ronald T. Raines, b and Younghoon Lee a, * a Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea b Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Received 12 January 2004 Abstract 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 IC 50 value of gONC was 50-fold lower than that of ONC. Glycosylation increased both the T m of ONC and its resistance to proteinase K, suggesting that the elevated cytotoxicity of gONC is related to higher conformational stability. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Onconase; Cancer chemotherapeutic agent; Glycosylation; Conformational stability; Cytotoxicity; Pichia pastoris expression system Onconase (ONC) isolated from oocytes or early em- bryos of Rana pipiens is selectively cytotoxic to cancer cells, both in vitro and in vivo [1,2]. The amino acid sequence of ONC displays 30% identity to that of ri- bonuclease A (RNase A) [3]. Although the active site and tertiary structure of ONC are similar to those of RNase A, ONC has a more compact structure. ONC is 20 residues shorter than RNase A, whereby all the de- letions are positioned in surface loops and at the N- terminus [4]. The protein has been evaluated in Phase I and II human clinical trials for the treatment of nu- merous solid tumors [5,6] and is currently in Phase III human clinical trials for malignant mesothelioma ther- apy. ONC-treated cells display apoptosis associated with cell blebbing, nuclear pyknosis, DNA fragmenta- tion, PARP (polyADP-ribose polymerase) cleavage, and activation of caspase 3-like activity [7]. Ribonucleolytic activity is required for ONC cytotoxicity [7–10]. In ad- dition to anti-cancer activity, the protein has anti-viral properties. At 10 8 M, ONC inhibits HIV-1 replication in chronically infected human cells by degrading viral RNA [11,12]. Pancreatic ribonucleases, such as RNase A, are inhibited by ribonuclease inhibitor (RI), a 50-kDa protein that binds to ribonucleases with 1:1 stoichiom- etry and dissociation constants below 1 pM [13–15]. Since ONC is not inhibited by RI [16], it can catalyze RNA degradation within the cell. ONC contains a disulfide bond between Cys87 and Cys104, which links the C-terminal Cys104 residue to a central b-strand. This Cys87–Cys104 disulfide bond is unique to the amphibian homologs of RNase A. Re- moval of the bond results in a significant decrease in conformational stability and cytotoxic activity [17–19]. The conformational stability of ribonucleases correlates directly with cytotoxicity and resistance to proteolysis [20,21], and could thus be a key determinant of ONC cytotoxicity by facilitating escape from proteolysis. The non-native Cys4–Cys118 disulfide bond of pancreatic * Corresponding author. Fax: +82-42-869-2810. E-mail address: Younghoon.Lee@kaist.ac.kr (Y. Lee). 1 Present address: Sensory Research Center, National Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Korea. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.153 Biochemical and Biophysical Research Communications 315 (2004) 976–983 BBRC www.elsevier.com/locate/ybbrc