Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture

Jung, Jae-Wan

doi:10.1007/s11816-022-00757-x

Cited by 3 publications

(4 citation statements)

References 43 publications

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“…For 100 mg of mass, 200 µL of 1X phosphate-buffered saline was added and homogenized by using a pestle tip. Mutant rice had T-DNA insertion on α-mannosidase, which is an enzyme mediating early stage of N-glycosylation in plants and showing down-regulated levels of plant-speci c N-glycans, was used as positive control (Jung, 2022). Protein extracts (30 µL/ sample) were separated via 12 % SDS-polyacrylamide gel electrophoresis and stained using Coomassie brilliant blue (SDS-PAGE).…”

Section: Sds-page and Western Blot Analysis Against To Plant N-glycansmentioning

confidence: 99%

“…Separation and identi cation of N-glycans from rice total glycoprotein were conducted as described in previous studies with a slight modi cation (Jung, 2022; Y. J. Shin et al, 2011).…”

Section: N-glycan Analysis Using Mass Spectrophotometrymentioning

confidence: 99%

“…3D-02972 and 1C-03248 both had a T-DNA insertion on Os06g0229200 and showed dramatically reduced Le a epitope detection while wild type and other GalT candidates showed strong multiple bands. A positive control, α-mannosidase I mutant have T-DNA insertion in the ER/Golgi-α-mannosidase I, which plays a role in very early stage of N-glycosylation, showed bands that are assumed as a result of insu cient knock-out by T-DNA insertion on the intron or minor activity of α-mannosidase I isotype (Jung, 2022). From this result, Os06g0229200 seems have important functions in the galactosylation of N-glycans in rice.…”

Section: Screening Of Mutant Contains Reduced Le a Epitopesmentioning

confidence: 99%

“…Beihammer, Maresch, Altmann, Van Damme,& Strasser, 2021). Indeed, because two knock-out mutant rice of Nacetylglucosaminyltransferase I and α-mannosidase I showed secretion of heterologous recombinant proteins into culture media in previous studies(Jung, 2022;Jung et al, 2019;Jung et al, 2017), secretion would probably not entirely depend on its N-glycan pattern. Further studies, such as comparing the level of secretion of a single protein in the GalT over expressing cell line with the Os06g0229200 or observation of Os06g0229200 under certain stress conditions, are expected to answer what is the exact functions of Le a epitopes.The decrease in α1,4-fucose on the Os06g0229200 line was expected due to the substrate speci city of α1,4-fucosyltransferase(Leonard et al, 2002;Leonard et al, 2005; Strasser et al, 2007;Wilson et al, …”

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confidence: 99%

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β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa

Jung

Kim

2023

Transgenic Res

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β1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of β1,3-galactose and α1,4-fucose by individual β1,3galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan pro ling using matrix-associated laser desorption/ionization-time of ight mass spectrometry (MALDI-TOF). Identi ed N-glycan species containing β1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.

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Section: Sds-page and Western Blot Analysis Against To Plant N-glycansmentioning

confidence: 99%

“…Separation and identi cation of N-glycans from rice total glycoprotein were conducted as described in previous studies with a slight modi cation (Jung, 2022; Y. J. Shin et al, 2011).…”

Section: N-glycan Analysis Using Mass Spectrophotometrymentioning

confidence: 99%

Section: Screening Of Mutant Contains Reduced Le a Epitopesmentioning

confidence: 99%

mentioning

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β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa

Jung

Kim

2023

Transgenic Res

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

Harvey

2024

Mass Spectrometry Reviews

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The use of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates is a well‐established technique and this review is the 12th update of the original article published in 1999 and brings coverage of the literature to the end of 2022. As with previous review, this review also includes a few papers that describe methods appropriate to analysis by MALDI, such as sample preparation, even though the ionization method is not MALDI. The review follows the same format as previous reviews. It is divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of computer software for structural identification. (2) Applications to various structural types such as oligo‐ and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other general areas such as medicine, industrial processes, natural products and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. MALDI is still an ideal technique for carbohydrate analysis, particularly in its ability to produce single ions from each analyte and advancements in the technique and range of applications show little sign of diminishing.

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β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N- glycan in Oryza sativa

Jung

Kim

2022

Preprint

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β1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of β1,3-galactose and α1,4-fucose by individual β1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing β1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.

show abstract

Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture

Cited by 3 publications

References 43 publications

β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa

β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

β1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N- glycan in Oryza sativa

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