Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants

Naphatsamon Uthailak; Hiroyuki Kajiura; Ryo Misaki; Kazuhito Fujiyama

doi:10.3389/fpls.2021.683762

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants

Front Plant Sci. 2021 Jun 7:12:683762. doi: 10.3389/fpls.2021.683762. eCollection 2021.

Authors

Naphatsamon Uthailak¹, Hiroyuki Kajiura^{1

2}, Ryo Misaki^{1

2}, Kazuhito Fujiyama^{1

2

3}

Affiliations

¹ International Center for Biotechnology, Osaka University, Osaka, Japan.
² Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan.
³ Cooperative Research Station in Southeast Asia, International Center for Biotechnology, Osaka University, Mahidol University, Bangkok, Thailand.

Abstract

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

Keywords: N-acetylglucosaminyltransferase I; N-glycosylation; Nicotiana benthamiana; plant-made pharmaceuticals; transient expression; β-glucocerebrosidase.