N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells

Misaki, Ryo; Sakai, Yohei; Ômasa, Takeshi; Fujiyama, Kazuhito; Seki, Tatsuji

doi:10.1016/j.jbiosc.2011.07.002

Cited by 9 publications

(7 citation statements)

References 43 publications

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“…In addition, in carrot cells, glucocerebrosidase fused to Ct‐VSS from tobacco chitinase A has mainly paucimannosidic structures (Shaaltiel et al ., ), supporting trimming of GlcNAc residues in vacuoles. Also, mouse IgG fused to the sporamin NPIRL ssVSS expressed in tobacco BY2 cells had mainly MMXF oligosaccharide (Misaki et al ., ). At differences of these reports, we did not detect paucimannosidic structures in vac‐Abs produced in tobacco leaves.…”

Section: Discussionmentioning

confidence: 97%

“…hexosaminidases) (Liebminger et al, 2011) and the presence of significant amounts of MMXF structures in the plant apoplastic fluid (Schneider et al, 2015) point to a broader distribution. Interestingly, the expression of a vacuolar sorted IgG in tobacco BY2 cells showed an increased level of MMXF structures when both heavy chain (HC) and light chain (LC) are fused to sporamin ssVSS in comparison with when only HC is fused (Misaki et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Ocampo

Lareu

Viegas

et al. 2016

Plant Biotechnology Journal

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SummaryPlant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Ocampo

Lareu

Viegas

et al. 2016

Plant Biotechnology Journal

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“…The plant vacuole is one of the largest subcellular compartments that storage ions and metabolites ( Marty, 1999 ). Although it is considered a hostile environment for foreign protein accumulation ( Hood et al, 2014 ), some proteins accumulate at high levels in central vacuoles such as glucocerebrosidase in carrot cells ( Shaaltiel et al, 2007 ), IgG in tobacco BY2 cells ( Misaki et al, 2011 ), human alpha-mannosidase in tobacco leaves ( De Marchis et al, 2013 ), human complement factor C5a in both N. tabacum and N. benthamiana leaves ( Nausch et al, 2012 ) and human collagen in tobacco leaves ( Stein et al, 2009 ). Other proteins such as human IgG1 and G4 have higher apo yield compared to the accumulation in ER and vacuoles in carrot suspension cell cultures ( Shaaltiel et al, 2006 ).…”

Section: Discussionmentioning

confidence: 99%

Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

et al. 2015

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Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.

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“…Agrobacterium carrying a plant expression vector were inoculated onto 23YT medium containing antibiotics (50 mg/L kanamycin, 200 mg/L streptomycin, and 50 mg/L rifampicin) to transform tobacco BY2 cells by the Agrobacterium-mediated transformation method (16). The transformants were selected on mLS medium (17,18) containing antibiotics (250 mg/L carbenicillin sodium salt and 150 mg/L kanamycin).…”

Section: Methodsmentioning

confidence: 99%

Cloning of a cDNA Encoding the Gly m Bd 28K Precursor and Its Vacuole Transport in Tobacco BY2 Suspension-Cultured Cells

Yumioka-Ito

Misaki

Yokoro

et al. 2014

J Nutr Sci Vitaminol

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Summary Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5′-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/ BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form. Key Words soybean allergen, Gly m Bd28K, 23-kDa peptide, signal peptide, vacuole E-mail: yumioka@icb.osaka-u.ac.jp The new nucleotide sequence of the 5′-nucleotide sequence region of cDNA encoding the Gly m Bd 28K precursor elucidated in the present study was registered under accession number AB046874.2 on DDBJ.

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N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells

Cited by 9 publications

References 43 publications

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

Cloning of a cDNA Encoding the Gly m Bd 28K Precursor and Its Vacuole Transport in Tobacco BY2 Suspension-Cultured Cells

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