Characterization and downstream mannose phosphorylation of human recombinant α‐L‐iduronidase produced in Arabidopsis complex glycan‐deficient (cgl) seeds. Issue 9 (31st July 2013)

• Record Type: Journal Article
• Authors: He, Xu; Pierce, Owen; Haselhorst, Thomas; von, Mark; Kolarich, Daniel; Packer, Nicolle H.; Gloster, Tracey M.; Vocadlo, David J.; Qian, Yi; Brooks, Doug; Kermode, Allison R.
• Is Part Of: Plant biotechnology journal. Volume 11:Issue 9(2013:Dec.)
• Page Start: 1034
• Page End: 1043
• Publication Date: 2013-07-31
• Subjects: Plant biotechnology -- Periodicals; Plant genetic engineering -- Periodicals; 630.272
• DOI: 10.1111/pbi.12096
• Languages: English
• ISSNs: 1467-7644

Abstract

<abstract abstract-type="main" id="pbi12096-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="pbi12096-sec-0001" sec-type="section"> <title>Summary</title> <p>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. <italic>Arabidopsis cgl</italic> mutants are characterized by a deficiency of the activity of <italic>N</italic>‐acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex <italic>N</italic>‐glycan biosynthesis. To develop a seed‐based platform for the production of recombinant IDUA for potential treatment of MPS I, <italic> cgl</italic> mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the <italic>N</italic>‐linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that <italic>cgl</italic>‐IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme<sup>TM</sup>). The <italic>N</italic>‐glycan profile showed that <italic>cgl</italic>‐derived IDUA contained predominantly high‐mannose‐type <italic>N</italic>‐glycans (94.5%), and the residual complex/hybrid <italic>N</italic>‐glycan‐containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified <italic>cgl</italic>‐IDUA was amenable to sequential <italic>in vitro</italic> 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 <italic>N</italic>−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−<italic>N</italic>−acetylglucosaminidase (the 'uncovering enzyme'). <italic>Arabidopsis</italic> 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.</p> </sec> </abstract>