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

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.

Impact of Specific N-Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays

“…Plant complex N -glycans can be further elongated by the incorporation of β1,3-galactose and α1,4-fucose, which results in the generation of Lewis A containing N -glycans ( Strasser et al, 2007 ). While this complex N -glycan modification is only found on a small number of native plant glycoproteins ( Beihammer et al, 2021 ), substantial amounts of Lewis A structures can be present on moss- or plant-produced recombinant glycoproteins such as human erythropoietin or β-glucocerebrosidase ( Parsons et al, 2012 ; Castilho et al, 2013 ; Uthailak et al, 2021 ). To examine whether human sera harbor antibodies directed against Lewis A containing N -glycans, we generated recombinant RBD-215 modified with Lewis A structures on both N -glycans.…”

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“…Suspension cultures help to reduce different types of polypeptides, resulting in a decreased variety of types and sizes of cells 15 . The plant cell suspension is a fast, simple, and cheaper technique where the cultures produce biomedicine 16,17 . Despite its applications, this method falls with a drawback of feedback inhibition as the product obtained using cell suspension culture will limit recombinant proteins 18 .…”

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects

“…Another inhibitor, swainsonine, has been used as additive in transgenic rice cultures to generate Man5 N-glycans by inhibition of α-mannosidase II (Choi et al, 2018). Man5 structures can also be created through the use of a N-acetyl-glucosaminyltransferase (GnT) I RNAi line of N. benthamiana, but this strategy also seemed to result in shorter Man3 and Man4 structures (Uthailak et al, 2021). Engineering toward specific mannosidic glycans remains challenging, as the necessary glyco-engineering strategies have not yet been developed or require additional optimization.…”