Production and Enzyme Engineering of Human Acetylcholinesterase and Its Mutant Derivatives

摘要

Design of bio-scavengers of organophosphate (OP) agents based on the mold of recombinant human acetyicholinesterase (rHuAChE), was carried out by two complementing research efforts: a. structure- reactivity characteristics of RHuAChE and of its mutant derivatives with various ligands b. Analysis of postranslation modifications responsible for the limited residence time of RHuAChE. The contribution of residues comprising the acyl-pocket, H-bond network, alkoxy pocket, oxyanion hole as well as the peripheral anionic subsite to interaction of OP agents in general, and specifically of nerve agents (such as soman, sarin and VX) in processes of phophonylation, aging and reactivation was probed by mutagenesis , kinetic studies, x-ray crystallography and mass spectrometry. These studies allowed for the delineation and selection of the key target residues for engineering of an optimal OP-scavenger into the human AChE template. Analysis of glycan composition of rHuAChE indicates that this enzyme is undersialylated and that this deficiency is a key parameter determining the pharmacological behavior of the recombinant enzyme. To overcome this limitation, we have genetically manipulated HEK-293 cells to produce high levels of both golgi-bound 2,6 sialyltransferase and of HuAChE. The recombinant AChE produced in these cells displays high sialic acid contents and is retained in the circulation for considerably longer periods of time than enzyme from non- manipulated cells. Taken together, these studies underline the role of post translation modification in AChE pharmacokinetics and demonstrate the ability to modulate enzyme biogenesis by genetic manipulation of the glycosylation machinery.