Tailoring structure–function properties of L-asparaginase: engineering resistance to trypsin cleavage

Abstract

Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.

Structure-Function Relationships and Clinical Applications of L-Asparaginases

“…In a recently published work an efficient approach for improving thermal and protease stability of EwA based on the combination of genetic engineering and site-specific chemical modification techniques was described [58]. The sensitivity of EwA to protease (e.g.…”

“…trypsin) inactivation and its low half-life has hindered its development as a clinical therapeutic. To develop an improved form of EwA, a structure-based molecular engineering approach was applied in combination with chemical modification (PEGylation) [58]. Limited proteolysis of EwA with trypsin was found to be associated with a cleavage first of the peptide bond between Lys53 and Gly54, accompanied by a second cleavage at the Arg206-Ser207 position of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206.…”

Purification and immobilization of l-arginase from thermotolerant Penicillium chrysogenum KJ185377.1; with unique kinetic properties as thermostable anticancer enzyme

“…Consistently, shielding of the surface proteolytic recognition sites on the target enzyme by PEGylation was extensively reported (Kotzia et al 2007;Yang et al 2004a).…”

Biochemical and Pharmacokinetic Properties of Aspergillus flavipes Glutathione-Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor

“…Upon trypsinolysis, the PEG-GHTHase retains more than 85% of its initial activity comparing to less than 10 % to free GHTHase. Masking of the surface recognition proteolytic sites by PEGylation was one of the main practical affordable benefits, to increasing the enzymes half-life time by decreasing their proteolysis, in vivo [1,26,33]. Higher induction of trypsin levels in blood plasma is a noticeable biochemical response to almost of enzymes dependent therapies [34].…”

“…Higher induction of trypsin levels in blood plasma is a noticeable biochemical response to almost of enzymes dependent therapies [34]. Practically, trypsin usually attacks the peptide bonds at recognition sites Lys [33]. Since, we utilized modified PEG residues, specifically to makes conjugates with surface lysine, thus the extending of enzyme activity and relative structural stability against could be justified to the inaccessibility to lysine residues to cleavage by trypsin [35].…”