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

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

doi:10.1111/pbi.12096

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 (Aldurazyme™). 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.

Highlights

Lysosomal storage diseases (LSDs) collectively represent over 70 inherited metabolic disorders
Mucopolysaccharidosis I (MPS I) disease is an LSD characterized by the deficiency of a-L-iduronidase (IDUA), an enzyme involved in the stepwise degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate
We address the three challenges associated with plant-based platforms for the production of recombinant lysosomal enzymes destined for Enzyme replacement therapy (ERT), focussing on human IDUA

Summary

Introduction

Lysosomal storage diseases (LSDs) collectively represent over 70 inherited metabolic disorders. Enzyme replacement therapy (ERT) is the conventional treatment. While some enzyme replacement therapeutics are in the ‘pipeline’ and are under different phases of clinical evaluation (van Gelder et al, 2012), to date, these therapeutics have been registered for only a few of the LSDs: Gaucher disease, Fabry disease, Pompe disease and mucopolysaccharidoses (MPS I, II, and VI). ERT is a process that takes advantage of plasma membrane receptor uptake to deliver recombinant enzyme into a patient’s cells following intravenous infusion of the replacement enzyme (Neufeld, 2011). Mucopolysaccharidosis I (MPS I) disease is an LSD characterized by the deficiency of a-L-iduronidase (IDUA), an enzyme involved in the stepwise degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. The current approved ERT for MPS I (AldurazymeTM or Laronidase), based on the recombinant IDUA from Chinese hamster ovary (CHO) cells, is prohibitively expensive, costing approximately US$450 000 per year for an average 12-year-old child.

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Discussion

Conclusion