Expression of β-1,4-galactosyltransferase and suppression of β-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells

Kim, Yeon Kyu; Kim, Kyoung Ro; Kang, Dong Gyun; Jang, So Young; Kim, Young Hwan; Joon, Hyung

doi:10.1016/j.jbiotec.2011.03.021

Cited by 22 publications

(8 citation statements)

References 26 publications

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“…By using RNAi and chemical inhibitors, the patterns of N ‐glycosylation in insect cells can be changed from paucimannose N ‐glycans to complex N ‐glycan structures with terminal GlcNAc and/or galactose. The RNAi approach was used to inhibit the GlcNAcase activity of DmFDL and SfFDL by using DmFDL/SfFDL‐specific double‐stranded RNA in Drosophila S2 cells and Sf9 cells (Geisler et al, ; Kim et al, ). The inhibitor 2‐ADN (2‐acetamido‐1,2‐dideoxynojirimycin) has been used to suppress the GlcNAcase activity to change the N ‐glycan patterns in S2 cells (Kim et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…The α‐mannosidase II removes the two terminal mannosyl residues from the α1,6‐branch, and then the N ‐acetylglucosaminyl transferase II (GlcNAcT II) adds a GlcNAc‐2 residue to the α1,6‐antenna. The activity of the GlcNAcT II is strictly dependent on the presence of GlcNAc‐1 added by the GlcNAcT I (Altmann and Marz, ; Kim et al, ; Kim et al, ). The insect FDL encoded by the gene FDL functions in the removal of the GlcNAc‐1.…”

Section: Introductionmentioning

confidence: 99%

“…The insect FDL encoded by the gene FDL functions in the removal of the GlcNAc‐1. Thus, when FDL is functional, subsequent enzymes in the pathway, such as GlcNAcT II, GalT, or SiaT are ineffective, resulting in a simple paucimannosidic N ‐glycan structure without galactosylation and further chain elongations (Altmann et al, ; Boquet et al, ; Kim et al, ; Kim et al, ). The DmFDL obtained from Drosophila melanogaster is the first FDL to be characterized (Leonard et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Biochemical studies indicate that both DmFDL and SfFDL have the specificity to hydrolyze the terminal β‐1,2‐GlcNAc residue from the α‐1,3 branch instead of the α‐1,6 branch of the substrate GnGn‐PA (Leonard et al, ; Geisler et al, ). In Drosophila S2 cells, when the activity of the GlcNAcase FDL is suppressed by RNAi or chemical inhibitors, the N ‐glycan structures were altered from the paucimannosidic core N ‐glycans to a complex‐type N ‐glycan (Kim et al, ; Kim et al, ).…”

Section: Introductionmentioning

confidence: 99%

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BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

Huo

Chen

et al. 2013

Arch Insect Biochem Physiol

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The β-N-acetylhexosaminidase FDL specifically removes the β-1,2-GlcNAc residue conjugated to the α-1,3-mannose residue of the core structure of insect N-glycans, playing significant physiological roles in post-translational modification in the Golgi apparatus. Little is known about its enzymatic properties. We obtained the OfFDL gene from the insect Ostrinia furnacalis by RT-PCR. The full length cDNA of FDL is 2241 bp carrying an opening reading frame of 1923 bp encoding 640 amino acids. The recombinant protein OfFDL in a soluble and active form was obtained with high purity through a two-step purification strategy. The recombinant OfFDL exclusively hydrolyzes the terminal β-1,2-GlcNAc residue from the α-1,3 branch instead of the α-1,6 branch of the substrate GnGn-PA. Several kinetic parameters including kcat/Km values toward four artificial substrates and Ki values of three representative hexosaminidase inhibitors were obtained.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

Huo

Chen

et al. 2013

Arch Insect Biochem Physiol

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show abstract

“…The terminal GlcNac glycans constituted up to 11% of the total glycoforms detected [81]. Concomitant expression of b-1,4-galactosyltransferase and suppression of b-N364 acetylglucosaminidase led to 22% of the glycans being in the form of a single-antennae extended to terminal Gal, with the other antennae not extended and ending in annose [82]. However, the main glycoform remained as paucimannose, while bi-antennary terminal Gal glycans were also detected in very low amounts.…”

Section: S2 Cell Glycosylationmentioning

confidence: 99%

The use ofDrosophilaS2 cells in R&D and bioprocessing

Jongh¹,

Salgueiro²,

Dyring³

2013

Pharmaceutical Bioprocessing

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Drosophila Schneider 2 (S2) cells have been available for approximately 40 years. Their use has intensified over the past 15 years: resolution of the whole Drosophila melanogaster genome and the amenability of S2 cells for siRNA-based studies are some of the reasons for their growing use. This review covers recent publications on use of S2 cells for research and manufacturing and points to some possible future developments in their use in the vaccine field. Relatively few groups have systematically developed the system to enable expression of challenging proteins. They demonstrated that these cells can constitute a robust, efficient protein expression system, with specific advantages such as homogeneous glycosylation profile; reproducibility between production runs; options for cultivation modes, including perfusion; and no cell lysis, leading to relatively low levels of contaminating host cell proteins. The platform has shown to be particularly well adapted for the production of challenging viral, malaria and immunotherapy antigens.

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Glycosylation in Drosophila S2 cells

Xu,

Tong,

Zhang

et al. 2024

Biotech & Bioengineering

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In recent years, there has been a remarkable surge in the approval of therapeutic protein drugs, particularly recombinant glycoproteins. Drosophila melanogaster S2 cells have become an appealing platform for the production of recombinant proteins due to their simplicity and low cost in cell culture. However, a significant limitation associated with using the S2 cell expression system is its propensity to introduce simple paucimannosidic glycosylation structures, which differs from that in the mammalian expression system. It is well established that the glycosylation patterns of glycoproteins have a profound impact on the physicochemical properties, bioactivity, and immunogenicity. Therefore, understanding the mechanisms behind these glycosylation modifications and implementing measures to address it has become a subject of considerable interest. This review aims to comprehensively summarize recent advancements in glycosylation modification in S2 cells, with a particular focus on comparing the glycosylation patterns among S2, other insect cells, and mammalian cells, as well as developing strategies for altering the glycosylation patterns of recombinant glycoproteins.

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Expression of β-1,4-galactosyltransferase and suppression of β-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells

Cited by 22 publications

References 26 publications

BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

The use ofDrosophilaS2 cells in R&D and bioprocessing

Glycosylation in Drosophila S2 cells

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