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dc.contributor.authorHe, Xu
dc.contributor.authorGalpin, Jason D
dc.contributor.authorTropak, Michael B
dc.contributor.authorMahuran, Don
dc.contributor.authorHaselhorst, Thomas
dc.contributor.authorvon Itzstein, Mark
dc.contributor.authorKolarich, Daniel
dc.contributor.authorPacker, Nicolle H
dc.contributor.authorMiao, Yansong
dc.contributor.authorJiang, Liwen
dc.contributor.authorGrabowski, Gregory A
dc.contributor.authorClarke, Lorne A
dc.contributor.authorKermode, Allison R
dc.date.accessioned2017-05-05T00:30:44Z
dc.date.available2017-05-05T00:30:44Z
dc.date.issued2012
dc.date.modified2014-08-28T05:07:51Z
dc.identifier.issn0959-6658
dc.identifier.doi10.1093/glycob/cwr157
dc.identifier.urihttp://hdl.handle.net/10072/42727
dc.description.abstractThere 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 ߭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.
dc.description.peerreviewedYes
dc.description.publicationstatusYes
dc.languageEnglish
dc.language.isoeng
dc.publisherOxford University Press
dc.publisher.placeUnited Kingdom
dc.relation.ispartofstudentpublicationN
dc.relation.ispartofpagefrom492
dc.relation.ispartofpageto503
dc.relation.ispartofissue4
dc.relation.ispartofjournalGlycobiology
dc.relation.ispartofvolume22
dc.rights.retentionY
dc.subject.fieldofresearchBiological sciences
dc.subject.fieldofresearchEnzymes
dc.subject.fieldofresearchReceptors and membrane biology
dc.subject.fieldofresearchStructural biology (incl. macromolecular modelling)
dc.subject.fieldofresearchBiomedical and clinical sciences
dc.subject.fieldofresearchBiochemistry and cell biology
dc.subject.fieldofresearchcode31
dc.subject.fieldofresearchcode310106
dc.subject.fieldofresearchcode310110
dc.subject.fieldofresearchcode310112
dc.subject.fieldofresearchcode32
dc.subject.fieldofresearchcode3101
dc.titleProduction of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan deficient (cgl) plants
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.date.issued2012
gro.hasfulltextNo Full Text
gro.griffith.authorvon Itzstein, Mark
gro.griffith.authorHaselhorst, Thomas E.
gro.griffith.authorKolarich, Daniel
gro.griffith.authorPacker, Nicki


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