The Production of Human β-Glucocerebrosidase in Nicotiana benthamiana Root Culture

Uthailak, Naphatsamon; Ohashi, Takao; Misaki, Ryo; Fujiyama, Kazuhito

doi:10.3390/ijms19071972

Cited by 12 publications

(12 citation statements)

References 33 publications

(56 reference statements)

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“…This enzyme is also related to plant physiology, including cell wall degradation and to the activation of both phytohormones and chemical compounds (Morant et al, 2008;Pankoke et al, 2013). Internal βglucosidases were also reported previously (Naphatsamon et al, 2018). Our results indicated that this system has the potential to be competitive for recombinant GCase production with high levels of biological activity.…”

Section: Discussionsupporting

confidence: 82%

“…M, PageRuler TM protein ladder; Crude, crude protein; Purified GCase, purified GCase from Phenyl 650M elution fractions 6 (E6); +, Cerezyme ® . studies: 65.5 U/mg TSP (Limkul et al, 2016), 24 U/mg TSP (He et al, 2012), 44.5 U/mg TSP (Limkul et al, 2015), and 81.4 U/mg TSP (Naphatsamon et al, 2018). On the other hand, the low GCase activity observed in the negative control was attributed to internal β-glucosidases produced in plants, which plays important roles in plant defense systems.…”

Section: Discussionmentioning

confidence: 97%

“…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: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 97%

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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“…Indeed, hairy roots of many different plant species have been utilized to produce various both secondary metabolites and recombinant proteins of pharmaceutical value at varying yields, such as enzymes (Woods et al., 2008), vaccines and hormones (Woffenden et al., 2008; Skarjinskaia et al., 2013), antibodies in different formats (Wongsamuth and Doran, 1997; Lonoce et al., 2016, 2018). Production of enzymes for replacement therapy of rare diseases was also reported (Rodriguez-Hernandez et al., submitted; Naphatsamon et al., 2018). Recombinant proteins produced in engineered hairy root cultures can be also secreted in the culture medium simplifying downstream purification processes (Guillon et al., 2006; Häkkinen et al., 2014).…”

Section: Introductionmentioning

confidence: 91%

Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures

Massa

Paolini²,

Marino

et al. 2019

Front. Plant Sci.

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Human papillomavirus (HPV) tumor disease is a critical public health problem worldwide, especially in the developing countries. The recognized pathogenic function of E5, E6, and E7 oncoproteins offers the opportunity to devise therapeutic vaccines based on engineered recombinant proteins. The potential of plants to manufacture engineered compounds for pharmaceutical purposes, from small to complex protein molecules, allows the expression of HPV antigens and, possibly, the regulation of immune functions to develop very specific therapies as a reinforcement to available nonspecific therapies and preventive vaccination also in developed countries. Among plant-based expression formats, hairy root cultures are a robust platform combining the benefits of eukaryotic plant-based bioreactors, with those typical of cell cultures. In this work, to devise an experimental therapeutic vaccine against HPV, hairy root cultures were used to express a harmless form of the HPV type 16 E7 protein (E7*) fused to SAPKQ, a noncytotoxic form of the saporin protein from Saponaria officinalis, that we had shown to improve E7-specific cell-mediated responses as a fusion E7*-SAPKQ DNA vaccine. Hairy root clones expressing the E7*-SAPKQ candidate vaccine were obtained upon infection of leaf explants of Solanum lycopersicum using a recombinant plant expression vector. Yield was approximately 35.5 μg/g of fresh weight. Mouse immunization with vaccine-containing crude extracts was performed together with immunological and biological tests to investigate immune responses and anticancer activity, respectively. Animals were primed with either E7*-SAPKQ DNA-based vaccine or E7*-SAPKQ root extract-based vaccine and boosted with the same (homologous schedule) or with the other vaccine preparation (heterologous schedule) in the context of TC-1 experimental mouse model of HPV-associated tumor. All the formulations exhibited an immunological response associated to anticancer activity. In particular, DNA as prime and hairy root extract as boost demonstrated the highest efficacy. This work, based on the development of low-cost technologies, highlights the suitability of hairy root cultures as possible biofactories of therapeutic HPV vaccines and underlines the importance of the synergic combination of treatment modalities for future developments in this field.

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“…The produced enzyme in the latter case does not contain any targeting signal to the vacuole. 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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The Production of Human β-Glucocerebrosidase in Nicotiana benthamiana Root Culture

Cited by 12 publications

References 33 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

Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics

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