Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan deficient (cgl) plants

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Title Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan deficient (cgl) plants
Author He, Xu; Galpin, Jason D.; Tropak, Michael B.; Mahuran, Don; Haselhorst, Thomas Erwin; von Itzstein, Mark; Kolarich, Daniel; Packer, Nicolle H.; Miao, Yansong; Jiang, Liwen; Grabowski, Gregory A.; Clarke, Lorne A.; Kermode, Allison R.
Journal Name Glycobiology
Year Published 2012
Place of publication United Kingdom
Publisher Oxford University Press
Abstract There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana cgl mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, IC50 of isofagomine (IFG), and thermal stability of the cgl-derived GCase were comparable to those of imiglucerase, a commercially-available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose-terminated. Additional purification was required to remove the ~15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including use of a null cgl mutant.
Peer Reviewed Yes
Published Yes
Alternative URI http://dx.doi.org/10.1093/glycob/cwr157
Volume 22
Issue Number 4
Page from 492
Page to 503
ISSN 1460-2423
Date Accessioned 2012-01-18
Language en_US
Research Centre Institute for Glycomics
Faculty Faculty of Science, Environment, Engineering and Technology
Subject Enzymes; Receptors and Membrane Biology; Structural Biology (incl Macromolecular Modelling)
URI http://hdl.handle.net/10072/42727
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1

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