Production of recombinant human acid β-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease

Jung, Jae-Wan; Choi, Hong-Yeol; Huy, Nguyen-Xuan; Park, Heajin; Kim, Ha Hyung; Yang, Moon-Sik; Kang, Seung‐Hoon; Kim, Dong-Il; Kim, Nan-Sun

doi:10.1016/j.pep.2019.02.014

Cited by 11 publications

(8 citation statements)

References 46 publications

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“…Our results indicated that this system has the potential to be competitive for recombinant GCase production with high levels of biological activity. Two different GCase purification strategies have been reported: hexa histidine-tag (His-tag) and a three-step purification method (Nam et al, 2017;Jung et al, 2019). However, the removal of his-tag and the multiple steps required for purification may increase the production cost.…”

Section: Discussionmentioning

confidence: 99%

“…After Taliglucerase alfa became the first plant-made pharmaceutical approved by the U.S. Food and Drug Administration in 2012, studies on the use of plants for pharmaceutical and non-pharmaceutical recombinant protein production have been increasing (Van Dussen et al, 2013 ; Tschofen et al, 2016 ). Many studies have focused on the production of recombinant GCase in plants including Arabidopsis thaliana (He et al, 2012 ), rice suspension culture (Nam et al, 2017 ; Jung et al, 2019 ), and Nicotiana benthamiana (Limkul et al, 2015 , 2016 ; Naphatsamon et al, 2018 ). However, some studies did not report the N -glycan structure of recombinant GCase (Limkul et al, 2015 ; Nam et al, 2017 ), whereas one study revealed the majority of N -glycan structure containing residues of plant-specific glycan (β1,2-xylose and α1,3-fucose) (Naphatsamon et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…The metabolic response to phytosterols varies among individuals (Jones, 2015). This can be due to genetic differences, for example in genes encoding ApoE and CyP7A1.…”

Section: Glucosylated Phytosterols and Medicinal Propertiesmentioning

confidence: 99%

“…Active GBA has recently also been produced in root cultures of Nicotiana tobacco (Naphatsamon et al, 2018). Finally, GBA enzyme with high mannose N-glycans (gnt1-GBA) was produced in mutant rice (Jung et al, 2019).…”

Section: Lysosomal Enzymes and Their Production In Plantsmentioning

confidence: 99%

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics

et al. 2020

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Plants contain numerous glycoconjugates that are metabolized by specific glucosyltransferases and hydrolyzed by specific glycosidases, some also catalyzing synthetic transglycosylation reactions. The documented value of plant-derived glycoconjugates to beneficially modulate metabolism is first addressed. Next, focus is given to glycosidases, the central theme of the review. The therapeutic value of plant glycosidases is discussed as well as the present production in plant platforms of therapeutic human glycosidases used in enzyme replacement therapies. The increasing knowledge on glycosidases, including structure and catalytic mechanism, is described. The novel insights have allowed the design of functionalized highly specific suicide inhibitors of glycosidases. These so-called activity-based probes allow unprecedented visualization of glycosidases cross-species. Here, special attention is paid on the use of such probes in plant science that promote the discovery of novel enzymes and the identification of potential therapeutic inhibitors and chaperones.

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“…A rice suspension line was derived from a gnt1 plant and used to produce the recombinant human acid αor β-glucosidase. Only mannosylated N-glycans were detected on the two recombinant proteins secreted in the culture medium, with a high proportion of Man5 (58% for α-glucosidase and 80% for β-glucosidase) (Jung et al, 2017(Jung et al, , 2019.…”

Section: Introductionmentioning

confidence: 99%

Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans

et al. 2021

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Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.

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Production of recombinant human acid β-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease

Cited by 11 publications

References 46 publications

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

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

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics

Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans

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