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N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-l-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I

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Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy.

Abstract

Functional recombinant human alpha-l-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man5 (cgl-IDUA) or Man6−8 (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K m) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases.

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Acknowledgements

We thank Dr. John Hopwood for the anti-IDUA antibodies and Dr. Stuart Kornfeld for the recombinant GlcNAc-1-phosphotransferase (α2β2). This work was funded by a Michael Smith Foundation for Health Research Senior Scholar Award to ARK and by a Canadian Glycomics Network for Centres of Excellence (GlycoNet) Grant 280013 (10.13039/501100009056).

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Authors and Affiliations

  1. Department of Biological Sciences, Simon Fraser University, 8888 University Dr., Burnaby, BC, V5A 1S6, Canada

    Owen M. Pierce, Grant R. McNair, Xu He & Allison R. Kermode

  2. International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Osaka, 565, Japan

    Hiroyuki Kajiura & Kazuhito Fujiyama

  3. Department of Biotechnology, College of Life Sciences, Ritsumeikan University, 1-1-1 Noji-hagashi, Kusatsu, Shiga, 525-8577, Japan

    Hiroyuki Kajiura

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  1. Owen M. Pierce
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  2. Grant R. McNair
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  3. Xu He
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  4. Hiroyuki Kajiura
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  5. Kazuhito Fujiyama
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  6. Allison R. Kermode
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Contributions

HK and KF performed the N-glycan analyses, and wrote the associated methods; OMP, XH, and GRM performed all other experiments and/or analyzed the data; ARK conceived the project, supervised experiments and wrote the article with contributions of authors GRM and OMP.

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Correspondence to Allison R. Kermode.

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Pierce, O.M., McNair, G.R., He, X. et al. N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-l-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I. Plant Mol Biol 95, 593–606 (2017). https://doi.org/10.1007/s11103-017-0673-x

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