<i>In vivo</i> deglycosylation of recombinant glycoproteins in tobacco <scp>BY</scp>‐2 cells

Herman, Xavier; Far, Johann; Peeters, Marie; Quinton, Loïc; Chaumont, François; Navarre, Catherine

doi:10.1111/pbi.14074

Cited by 4 publications

(1 citation statement)

References 55 publications

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“…Typically, viruses contribute to their virulence by manipulating or evading the host's immune response capabilities [28,29]. Herpesvirus glycoproteins gB, gH, gD, gJ, and gM are all connected via asparagine-linked glycosylation, which plays an essential role in their proper folding, transport, and immune evasion [9,[30][31][32][33][34]. However, the precise role of DPV gI glycosylation in the context of viral proliferation and virulence within its natural host is an area that requires further investigation.…”

Section: Introductionmentioning

confidence: 99%

N-glycosylation of envelope glycoprotein gI is crucial for the duck plague virus proliferation and virulence

Cheng,

Ning,

Wang

et al. 2024

Preprint

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Duck plague virus (DPV) causes highly pathogenic duck plague, and the envelope glycoprotein gI, as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. In this study, reverse genetics technology was utilized to specifically target the gI protein within the genome of the DPV. Four mutant strains of DPV with mutations in the N-glycosylation sites of the gI protein were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues (N69, N78, and N265) of gI are N-glycosylation sites, and Western blot analysis substantiated that the glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI results in the misfolding of the protein and its consequent retention in the endoplasmic reticulum (ER). For the subsequent intracellular transport of gI within duck embryonic fibroblasts (DEFs), the carrying effect of its interacting protein gE is essential. Compared to the parental virus, the mutated virus exhibits a reduced intercellular transmission capability to 66.3%. In ducks, the deglycosylation of gI significantly reduces the replication of DPV in vivo, thereby impairing the virulence of DPV. This research marks the first successful generation of an attenuated DPV strain through targeted mutations at the N-glycosylation sites. The findings provide a foundational understanding of DPV pathogenesis and offer a basis for the development of live attenuated vaccines against the disease.

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Section: Introductionmentioning

confidence: 99%

N-glycosylation of envelope glycoprotein gI is crucial for the duck plague virus proliferation and virulence

Cheng,

Ning,

Wang

et al. 2024

Preprint

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Enhanced efficacy of glycoengineered rice cell‐produced trastuzumab

Shin,

Oh,

Jang

et al. 2024

Plant Biotechnology Journal

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SummaryFor several decades, a plant‐based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant‐specific N‐glycans attached in plant‐based biopharmaceuticals has not been completely solved. To eliminate all plant‐specific N‐glycan structure, eight genes involved in plant‐specific N‐glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon‐optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co‐cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P‐TMab) was identical to that of TMab. The inhibitory effect of P‐TMab on the proliferation of the BT‐474 cancer cell line was significantly enhanced at concentrations above 1 μg/mL (****P < 0.0001). P‐TMab bound to a FcγRIIIa variant, FcγRIIIa‐F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P‐TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P‐TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT‐474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P‐TMab has a novel and enhanced efficacy compared to TMab.

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N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus

Ning,

Wang,

Cheng

et al. 2024

Vet Res

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Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

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In vivo deglycosylation of recombinant glycoproteins in tobacco BY‐2 cells

Cited by 4 publications

References 55 publications

N-glycosylation of envelope glycoprotein gI is crucial for the duck plague virus proliferation and virulence

N-glycosylation of envelope glycoprotein gI is crucial for the duck plague virus proliferation and virulence

Enhanced efficacy of glycoengineered rice cell‐produced trastuzumab

N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus

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