The production of human glucocerebrosidase in glyco‐engineered <i><scp>N</scp>icotiana benthamiana</i> plants

Limkul, Juthamard; Iizuka, Sayoko; Sato, Yohei; Misaki, Ryo; Ohashi, Takao; Ohashi, Takuma; Fujiyama, Kazuhito

doi:10.1111/pbi.12529

Cited by 38 publications

(43 citation statements)

References 47 publications

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“…The same has been described for human EPO-Fc and transferrin when transiently expressed in N. benthamiana (Castilho et al, 2011). These proteins carry mainly fully processed GlcNAc terminating complex the N-glycans of recombinantly expressed follicle stimulating hormone, recombinant glucocerebrosidase targeted to the apoplast or human lactoferrin (Dirnberger et al, 2001;He et al, 2012;Limkul et al, 2016;Samyn-Petit et al, 2003). Further studies are needed to identify the protein intrinsic features that lead to efficient processing by plant HEXOs.…”

Section: Discussionmentioning

confidence: 85%

“…By contrast, human A1AT harbours considerably amounts of paucimannosidic structures on all three N ‐glycosylation sites (Castilho et al ., ). A similar result was observed for the N ‐glycans of recombinantly expressed follicle stimulating hormone, recombinant glucocerebrosidase targeted to the apoplast or human lactoferrin (Dirnberger et al ., ; He et al ., ; Limkul et al ., ; Samyn‐Petit et al ., ). Further studies are needed to identify the protein intrinsic features that lead to efficient processing by plant HEXOs.…”

Section: Discussionmentioning

confidence: 99%

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Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

Shin

Castilho

Dicker

et al. 2016

Plant Biotechnology Journal

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SummaryPlants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco‐engineering facilitate the elimination of nonmammalian‐type glycosylation and introduction of missing pathways for customized N‐glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N‐glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N‐glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β‐hexosaminidases (HEXOs) that account for the formation of paucimannosidic N‐glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO‐deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core α1,3‐fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N‐glycans on plant‐produced human α1‐antitrypsin.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

Shin

Castilho

Dicker

et al. 2016

Plant Biotechnology Journal

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“…This has generated valuable knowledge of plant glycosylation including paucimannosylation decorating many glycoproteins of potential therapeutic or other commercial value (Gomord et al ., ; Rasala et al ., ; Levy‐Ontman et al ., ; Mathieu‐Rivet et al ., ; Strasser, ; Mathieu‐Rivet, Lerouge, & Bardor, ). These observations do not reflect the natural plant glycoproteome, but still provide insights into the glycosylation ‘capacity’ and the glycosylation machinery of plants, in particular, when complemented with genetic or chemical manipulation of enzymes in the glycosylation machinery (He et al ., ; Castilho et al ., ; Limkul et al ., ).…”

Section: Surveying Pmps Across the Eukaryotic Kingdoms And Phylamentioning

confidence: 97%

Human protein paucimannosylation: cues from the eukaryotic kingdoms

et al. 2019

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Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α-or β-mannosyl-terminating asparagine (N )-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue-and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and β1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi-and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N -acetyl-β-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N -acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue-and development-specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N -acetyl-β-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongs...

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“…Traditionally, GD is classified into three clinical phenotypes ISSN 2059-7983 capabilities must be employed, as exemplified by the existing ERT expression systems. In addition to these expression platforms, GBA production has been attempted in murine cells (Fabrega et al, 2000), COS-1 cells (Grabowski et al, 1989), seeds of the Arabidopsis thaliana plant (He et al, 2012), glycoengineered Nicotiana benthamiana plants (Limkul et al, 2016), Pichia pastoris (Sinclair & Choy, 2002) and baculoviral expression vector systems (BEVS; Martin et al, 1988;Sinclair et al, 2006;Sawkar et al, 2006). Such diversity demonstrates the current lack of consensus on a robust and economical platform for GBA production for nonclinical use.…”

Section: Introductionmentioning

confidence: 99%

A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis

Rowland¹,

Wu²,

Liu³

et al. 2020

Acta Crystallogr D Struct Biol

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The lysosomal glycoside hydrolase -glucocerebrosidase (GBA; sometimes called GBA1 or GC ase ) catalyses the hydrolysis of glycosphingolipids. Inherited deficiencies in GBA cause the lysosomal storage disorder Gaucher disease (GD). Consequently, GBA is of considerable medical interest, with continuous advances in the development of inhibitors, chaperones and activity-based probes. The development of new GBA inhibitors requires a source of active protein; however, the majority of structural and mechanistic studies of GBA today rely on clinical enzyme-replacement therapy (ERT) formulations, which are incredibly costly and are often difficult to obtain in adequate supply. Here, the production of active crystallizable GBA in insect cells using a baculovirus expression system is reported, providing a nonclinical source of recombinant GBA with comparable activity and biophysical properties to ERT preparations. Furthermore, a novel crystal form of GBA is described which diffracts to give a 0.98 Å resolution unliganded structure. A structure in complex with the inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro--d-glucopyranoside was also obtained, demonstrating the ability of this GBA formulation to be used in ligand-binding studies. In light of its purity, stability and activity, the GBA production protocol described here should circumvent the need for ERT formulations for structural and biochemical studies and serve to support GD research.

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The production of human glucocerebrosidase in glyco‐engineered Nicotiana benthamiana plants

Cited by 38 publications

References 47 publications

Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

Human protein paucimannosylation: cues from the eukaryotic kingdoms

A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis

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