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Characterization and downstream mannose phosphorylation of human recombinant α-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds

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Xu He, Owen Pierce, Thomas Haselhorst, Daniel Kolarich, Nicolle H. Packer, Tracey Gloster, David J. Vocadlo, Yi Qian, Doug Brooks, Allison R. Kermode

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Abstract

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (AldurazymeTM). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells—UDP-GlcNAc:lysosomal enzyme N−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−N−acetylglucosaminidase (the ‘uncovering enzyme’). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

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Original languageEnglish
Pages (from-to)1034–1043
JournalPlant Biotechnology Journal
Volume11
Issue number9
Early online date31 Jul 2013
DOIs
Publication statusPublished - Dec 2013

    Research areas

  • Arabidopsis cgl mutant, Mucopolysaccharidosis I, Human α-L-iduronidase, Mannose-6-phosphate recognition marker, N-glycosylation

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