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ISSN 1674-8018(Online)

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Prot Cell    2011, Vol. 2 Issue (1) : 41-47    https://doi.org/10.1007/s13238-011-1007-4      PMID: 21337008
COMMUNICATION
Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes
Bo-jiao Yin1,2, Ting Gao1, Nuo-yan Zheng1,2, Yin Li2, San-yuan Tang1, Li-ming Liang2, Qi XIE1()
1. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China; 2. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingang Road W, Guangzhou 510275, China
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Abstract

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 N-glycans 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 glyco-engineered 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.
Keywords BY2 cells      N-glycosylation      glycosyltransferase      RNA interference     
Corresponding Author(s): XIE Qi,Email:qxie@genetics.ac.cn   
Issue Date: 01 January 2011
 Cite this article:   
Bo-jiao Yin,Ting Gao,Nuo-yan Zheng, et al. Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes[J]. Prot Cell, 2011, 2(1): 41-47.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1007-4
https://academic.hep.com.cn/pac/EN/Y2011/V2/I1/41
Fig.1  Mutiple sequence alignment and diagram of RNAi constructs.
(A) Alignment of conserved motifs from β1,2-XylT protein sequences. (B) Alignment of conserved motifs from α1,3-FucT protein sequences. The aligned amino acid sequences are from the plants L. cv Bright Yellow 2 (Nt), (At) and (Os). Black blocks represent amino acid identity between all sequences and gray blocks between two or three sequences. Broken lines correspond to the gaps required for the alignment of these sequences. (C) The construction diagram of FXR. The amplified DNAs encoding hairpin RNA (RNAi) designed to target silencing of endogenous tobacco BY2 genes encoding α1,3-fucosyltransferase and β1,2-xylosyltransferase. RB and LB, right and left borders; 35S, cauliflower mosaic virus 35S promoters.
Fig.1  Mutiple sequence alignment and diagram of RNAi constructs.
(A) Alignment of conserved motifs from β1,2-XylT protein sequences. (B) Alignment of conserved motifs from α1,3-FucT protein sequences. The aligned amino acid sequences are from the plants L. cv Bright Yellow 2 (Nt), (At) and (Os). Black blocks represent amino acid identity between all sequences and gray blocks between two or three sequences. Broken lines correspond to the gaps required for the alignment of these sequences. (C) The construction diagram of FXR. The amplified DNAs encoding hairpin RNA (RNAi) designed to target silencing of endogenous tobacco BY2 genes encoding α1,3-fucosyltransferase and β1,2-xylosyltransferase. RB and LB, right and left borders; 35S, cauliflower mosaic virus 35S promoters.
Fig.2  Analysis of the protein xylosylation and fucosylation in FXR lines.
(A) Western blot analysis of total protein extracts from transgenic RNAi lines (FXR1-12). The presence/absence of fucose and xylose residues was determined with anti-HRP antiserum. Numbers indicated the different transformed lines. (B) Western blot analysis of total protein extracts from WT and FXR7 lines from late growth phase using anti-xylose (left panel) and anti-fucose (right panel).
Fig.2  Analysis of the protein xylosylation and fucosylation in FXR lines.
(A) Western blot analysis of total protein extracts from transgenic RNAi lines (FXR1-12). The presence/absence of fucose and xylose residues was determined with anti-HRP antiserum. Numbers indicated the different transformed lines. (B) Western blot analysis of total protein extracts from WT and FXR7 lines from late growth phase using anti-xylose (left panel) and anti-fucose (right panel).
Fig.3  MALDI-TOF mass spectrum of N-glycans isolated from endogenous proteins of WT (A) and FXR7 (B) cells.
Fig.3  MALDI-TOF mass spectrum of N-glycans isolated from endogenous proteins of WT (A) and FXR7 (B) cells.
Fig.4  Structures of high-mannose-type (Man-5 to Man-9) and complex-type (a to e) N-linked oligosaccharides extracted from wild-type BY2 cells.
Fig.4  Structures of high-mannose-type (Man-5 to Man-9) and complex-type (a to e) N-linked oligosaccharides extracted from wild-type BY2 cells.
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