Plant‐derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum‐retention signal is <i>N</i>‐glycosylated homogeneously throughout the plant with mostly high‐mannose‐type <i>N</i>‐glycans

Triguero, Ada; Cabrera, Gleysin; Cremata, José A.; Yuen, Chun-Ting; Wheeler, Jun X.; Ramírez, Nadia

doi:10.1111/j.1467-7652.2005.00137.x

Cited by 94 publications

(58 citation statements)

References 44 publications

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“…N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural forms (Lerouge, 1998). If the transgenic protein is directed toward and retained in the ER, then 80% of the total N-glycans attached to ER-resident proteins in the transgenic plant would be high-mannose-type N-glycans (Helenius and Aevi, 2001;Triguero, 2005). The use of the ER-retention signal has been proposed to restrict glycosylation of plant-derived antibodies or the vaccine to only highmannose-type N-glycans (Triguero, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…If the transgenic protein is directed toward and retained in the ER, then 80% of the total N-glycans attached to ER-resident proteins in the transgenic plant would be high-mannose-type N-glycans (Helenius and Aevi, 2001;Triguero, 2005). The use of the ER-retention signal has been proposed to restrict glycosylation of plant-derived antibodies or the vaccine to only highmannose-type N-glycans (Triguero, 2005). However, in this study, the effect of Fc and KDEL on the expression levels and glycosylation patterns could not be investigated since we did not include 5Aβ without Fc or 5Aβ-Fc.…”

Section: Discussionmentioning

confidence: 99%

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Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Kim¹,

Youm²,

Lee³

et al. 2014

HST

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Transgenic potato was generated to express recombinant 5 repeated β-amyloid (Aβ) peptides, potential antigens to be applied as a preventive accine for Alzheimer's disease using Agrobacterium mediated transformation. The Aβ peptides were fused to the human IgG Fc fragment enhancing protein and KDEL, which is the endoplasmic reticulum (ER) retention signal (5Aβ-FcK). The 5Aβ-FcK, was expressed under the control of the duplicated 35S promoter. PCR analysis confirmed the presence of the transgene in several transgenic potato lines. Southern blot analysis showed only a single gene copy number in transgenic line 22, whereas multiple gene copy numbers were shown for transgenic lines 31 and 44. Northern blot analysis showed that line 22 had stronger mRNA levels when compared to lines 31 and 44. Immunoblot analysis confirmed that the 5Aβ-FcK protein was expressed in the transgenic potato plant. These results indicate that 5Aβ fused to Fc can be expressed in potato plants.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Kim¹,

Youm²,

Lee³

et al. 2014

HST

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“…In our study, we also found that the deficiency of XylT and FucT did not affect the morphology, growth rate, or growth status of tobacco BY2 cells. In previous studies, several strategies have been adopted to minimize the structural differences observed in complex Nglycans of plants and mammals (Palacpac et al, 1999;Triguero et al, 2005). Cox et al reported that RNAi was a valuable tool to eliminate the expression of core α1,3-FucT and β1,2-XylT in the aquatic plant species L. minor (Cox et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

et al. 2011

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Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing β1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.

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“…4). The KDEL sequence was incorporated because ER retention is known to increase antibody expression in plants (43); it appeared to be functional, as glycosylation analyses (Table 1) revealed high-mannose modifications consistent with ER localization (10,26,29,51). High mannose content is known to be essential for mediating effector function (54).…”

Section: Discussionmentioning

confidence: 99%

“…Plant glycosylations differ from mammalian glycosylations in both sugar content and pattern, as they lack sialylations (47) and residues, such as ␤(1,4)-Gal and ␣(2,6)-NeuAc, that are often found on mammalian glycans (17,19). MAbs expressed in transgenic tobacco plants are typically glycosylated with highmannose carbohydrates (10,17,29,48,51); thus, a major concern regarding plant-produced MAbs is whether they can support the recruitment of effector functions required for therapeutic efficacy against pathogen targets, such as bacteria.…”

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confidence: 99%

A Human Anti-Pseudomonas aeruginosaSerotype O6ad Immunoglobulin G1 Expressed in Transgenic Tobacco Is Capable of Recruiting Immune System Effector Function In Vitro

McLean

Almquist

Niu

et al. 2007

Antimicrob Agents Chemother

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The production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with V H and V L sequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizes Pseudomonas aeruginosa serotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other V H and V L antigen-binding coding sequences. The plant-produced IgG1 was determined to have highmannose glycan content and to be capable of mediating opsonophagocytosis of P. aeruginosa serotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.

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Plant‐derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum‐retention signal is N‐glycosylated homogeneously throughout the plant with mostly high‐mannose‐type N‐glycans

Cited by 94 publications

References 44 publications

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

A Human Anti-Pseudomonas aeruginosaSerotype O6ad Immunoglobulin G1 Expressed in Transgenic Tobacco Is Capable of Recruiting Immune System Effector Function In Vitro

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