Abstract
L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.
Highlights
L-Asparaginase is a homo-tetramer enzyme that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia [1]
Cysteine-directed PEGylation was performed using thiol-maleimide chemistry [36], to target cysteine residues previously introduced by mutagenesis at positions A38 and T263
We report an alternative cysteine-directed conjugation strategy to PEGylate the therapeutic enzyme L-asparaginase at non-natural positions introduced by mutagenesis
Summary
L-Asparaginase is a homo-tetramer enzyme that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia [1]. Some types of blood cancers, e.g., acute lymphoblastic leukemia and non-Hodgkin lymphoma, need to acquire L-Asn from extracellular sources due to a deficiency in the expression of asparagine synthetase.
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