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

Naphatsamon Uthailak,Kazuhito Fujiyama,Hiroyuki Kajiura,Ryo Misaki

doi:10.3389/fpls.2021.683762

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.

Highlights

Gaucher disease, one of the most common lysosomal storage disorders, is caused by the mutation of the GBA1 gene, resulting in the defective activity of a lysosomal enzyme called glucocerebrosidase (GCase, β-glucosidase; EC: 3.2.1.45)
To produce recombinant GCase in N. benthamiana plants, 4-week-old plants were infiltrated with Agrobacterium mixture harboring GCase and p19 vectors at an OD600 of 0.5
The GCase produced in GnTI) downregulated Nicotiana benthamiana ( (gntI) plants showed higher GCase activity (134.3 ± 17.3 U/mg total soluble protein (TSP)) than that produced in WT plants (81.5 ± 6.9 U/mg TSP)

Summary

Introduction

One of the most common lysosomal storage disorders, is caused by the mutation of the GBA1 gene, resulting in the defective activity of a lysosomal enzyme called glucocerebrosidase (GCase, β-glucosidase; EC: 3.2.1.45). Enzyme replacement therapy (ERT) is the most effective treatment for Gaucher disease type 1; it involves intravenous infusions of exogenous recombinant GCase to patients (Siebert et al, 2014; Stirnemann et al, 2017; Boer et al, 2020). Three drugs are commercially available: Imiglucerase (Cerezyme R , Genzyme Corporation), Velaglucerase alfa (VPRIV R , Shire Plc), and Taliglucerase alfa (Elelyso R , Pfizer). These drugs are recombinant GCases produced in Chinese hamster ovary (CHO) cells, human fibroblasts, and carrot suspension cells, respectively. All are glycoproteins containing different N-glycan structures (Bennett and Fellner, 2018).

Methods

Results

Conclusion