A mutation in the gene encoding cathepsin B in a patient with strongly reduced metabolism asparaginase. (A) Pharmacokinetics of Erwinase serum levels indicate a strongly delayed metabolism of ASNase in this patient. The half-life (ln2/Kel) of 28.5 hours (normal 7-15 hours5 ) was calculated using best-fit nonparametric modeling of the data points. (B) DNA sequencing of the cathepsin B gene reveals a heterozygous deletion of a single codon (c.709_711delAAG) in DNA isolated from PBMCs and buccal cells of the patient. (C) Cathepsin B activity in lysates of EBV-transformed B cells of the patient (carrying the mutation) and age-matched controls (N = 5) was determined by measuring cleavage of the fluorescent substrate Ac-RR-AFC. The plot shows an average of 2 experiments with standard deviation. One of the control samples was set to 1, and all samples were correlated to this sample. Unpaired 2-tailed t test was used to determine significance. (D) ASNase was incubated in lysate of HEK293 cells expressing wild-type or mutant cathepsin B. After incubation, residual ASNase activity was assayed as described in the supplemental Methods section. Cathepsin inhibitor CA-074 was included in selected samples to confirm the contribution of cathepsin B in this degradation. The plot shows an average of 3 independent experiments with standard error of the mean. Analysis of variance statistical analysis was applied to test for significance.
