A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug l-asparaginase
Naina Patel, … , Paul A. Bates, Vaskar Saha
Naina Patel, … , Paul A. Bates, Vaskar Saha
Published June 8, 2009
Citation Information: J Clin Invest. 2009;119(7):1964-1973. https://doi.org/10.1172/JCI37977.
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Research Article Oncology

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug l-asparaginase

Abstract

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

Authors

Naina Patel, Shekhar Krishnan, Marc N. Offman, Marcin Krol, Catherine X. Moss, Carly Leighton, Frederik W. van Delft, Mark Holland, JiZhong Liu, Seema Alexander, Clare Dempsey, Hany Ariffin, Monika Essink, Tim O.B. Eden, Colin Watts, Paul A. Bates, Vaskar Saha

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Figure 7

Decreased enzymatic activity of the N24G mutant is explained by MD simulations of N24 interactions.

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Decreased enzymatic activity of the N24G mutant is explained by MD simul...
The hydrogen bond network around N24 in WT ASNase (A) and the N24G mutant (B) is shown. Residue T12 is colored according to atom types. Key distances (in Å) show position of the T12 hydroxyl group with respect to the active site. Residues N24 and Y25 are colored brown and blue, respectively; other residues of the active site are in purple. Two residues that belong to another monomer but form hydrogen bonds (red) with either N24 or Y25 are shown in orange (D281) and green (E283). MD simulation showing orientation of the T12 hydroxyl group with respect to all 4 active sites at each time point during simulation in native ASNase (C) and the N24G mutant (D). The black part of each graph represents the fraction when the T12 hydroxyl group is in correct orientation with respect to the 4 active sites. The red part of each graph represents the fraction when the T12 hydroxyl group is incorrectly oriented relative to the 4 active sites (methyl instead of the hydroxyl group pointing toward the center of the active site).