Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation

All clinically-used asparaginases convert L-asparagine (L-Asn) to L-aspartate (L-Asp) and L-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different se-quences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that AginasaTM share similar sequence to EcA2 from E. coli strain BL21(DE3), while LeuginaseTM has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between AginasaTM (64D and 252 T) and LeuginaseTM (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations be-tween these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immuno-genic silent inactivation.

Automated summary

This study investigated the impact of sequence variations between two variants of E. coli L-asparaginase (EcA2) on their biological function, stability, and interchangeability. Results showed that although these two enzymes had similar specific activity in vitro, they exhibited different behavior in vivo. Further analysis revealed that the amino acid differences between the two variants led to conformational changes, affecting surface accessibility and susceptibility to proteolysis.