Reduction of N-linked xylose and fucose by expression of rat β1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression

Karg, Saskia R.; Frey, Alexander D.; Kallio, Pauli T.

doi:10.1016/j.jbiotec.2010.01.005

Cited by 21 publications

(10 citation statements)

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“…Previous studies have shown that the expression of GnTIII in wild-type plants resulted in the attachment of a bisecting GlcNAc (Rouwendal et al 2007; Frey et al 2009; Karg et al 2010). However, as the native human GnTIII is very likely targeted to a medial-Golgi compartment overlapping mainly with FT, but also with N -acetylglucosaminyltransferase II (GnTII) and XT different incompletely processed glycoforms were generated (Rouwendal et al 2007; Frey et al 2009).…”

Section: Discussionmentioning

confidence: 99%

“…The unwanted microheterogeneity of N-glycosylation is a serious limitation of these approaches and indicates the importance of precise subcellular targeting of recombinantly expressed glycosyltransferases for efficient N -glycan processing. For example, targeting of GnTIII to early instead of medial/late Golgi compartments resulted in an increase in incompletely processed bisected hybrid-type structures in CHO cells and in plants (Ferrara et al 2006; Frey et al 2009; Karg et al 2010). Comparable studies that demonstrate the successful overexpression of N -acetylglucosaminyltransferase IV (GnTIV) or V (GnTV) in hosts suitable for the production of recombinant glycoproteins have not been described so far.…”

Section: Introductionmentioning

confidence: 99%

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N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Castilho¹,

Gattinger²,

et al. 2011

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Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Castilho¹,

Gattinger²,

et al. 2011

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“…Major efforts are currently undertaken for remodeling the N -glycosylation pathway of plants to obtain more mammalian-like glycoforms. This includes (i) the elimination of the endogenous plant-specific β1,2-xylosyltransferase and core α1,3-fucosyltransferase activities (18, 29–31); (ii) elongation of N -glycans with β1,4-galactose residues (32–34); (iii) attachment of a bisecting GlcNAc (35–37); (iv) the generation of core α1,6-fucosylated N -glycans (38); and (v) finally the generation of sialylated glycoforms (39). In this study, we established that two β- N -acetylhexosaminidases, HEXO1 and HEXO3, account for the conversion of complex into paucimannosidic N -glycans in Arabidopsis .…”

Section: Discussionmentioning

confidence: 99%

β-N-Acetylhexosaminidases HEXO1 and HEXO3 Are Responsible for the Formation of Paucimannosidic N-Glycans in Arabidopsis thaliana

Liebminger¹,

Veit²,

Pabst³

et al. 2011

Journal of Biological Chemistry

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Most plant glycoproteins contain substantial amounts of paucimannosidic N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. We now demonstrate that two β-N-acetylhexosaminidases (HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. Total N-glycan analysis of hexo knock-out plants revealed that HEXO1 and HEXO3 contribute equally to the production of paucimannosidic N-glycans in roots, whereas N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Because hexo1 hexo3 double mutants do not display any obvious phenotype even upon exposure to different forms of abiotic or biotic stress, it should be feasible to improve the quality of glycoprotein therapeutics produced in plants by down-regulation of endogenous β-N-acetylhexosaminidase activities.

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“…It is also known that glycosylation in plants differs from mammalian cells, especially glycosylation processed through Golgi apparatus. Many efforts were made to control the glycosylation of recombinant proteins in plants, such as knockin or knockout of related enzymes and retention of the target protein in ER [33]–[35]. In our case, fusion EiP was deposited in ER-derived protein body, thus no further modification would come from Golgi, which may provide an alternative way to produce recombinant proteins in transgenic plants.…”

Section: Discussionmentioning

confidence: 91%

A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform

Tian

Sun

2011

PLoS ONE

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BackgroundPlant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor.Methodology/Principal FindingsTo tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2–4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein.Conclusion/SignificanceThis study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.

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Reduction of N-linked xylose and fucose by expression of rat β1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression

Cited by 21 publications

References 50 publications

N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

β-N-Acetylhexosaminidases HEXO1 and HEXO3 Are Responsible for the Formation of Paucimannosidic N-Glycans in Arabidopsis thaliana

A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform

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