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A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminylt...

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Autores principales: Mabashi-Asazuma, Hideaki, Jarvis, Donald L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689212/
https://www.ncbi.nlm.nih.gov/pubmed/34838817
http://dx.doi.org/10.1016/j.jbc.2021.101454
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author Mabashi-Asazuma, Hideaki
Jarvis, Donald L.
author_facet Mabashi-Asazuma, Hideaki
Jarvis, Donald L.
author_sort Mabashi-Asazuma, Hideaki
collection PubMed
description Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN(Lec1). We found that Sf-RVN and Sf-RVN(Lec1) cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN(Lec1) cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN(Lec1) cells were Endo H-cleavable Man(5)GlcNAc(2) structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.
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spelling pubmed-86892122021-12-30 A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans Mabashi-Asazuma, Hideaki Jarvis, Donald L. J Biol Chem Methods and Resources Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN(Lec1). We found that Sf-RVN and Sf-RVN(Lec1) cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN(Lec1) cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN(Lec1) cells were Endo H-cleavable Man(5)GlcNAc(2) structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography. American Society for Biochemistry and Molecular Biology 2021-11-26 /pmc/articles/PMC8689212/ /pubmed/34838817 http://dx.doi.org/10.1016/j.jbc.2021.101454 Text en © 2021 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Methods and Resources
Mabashi-Asazuma, Hideaki
Jarvis, Donald L.
A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title_full A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title_fullStr A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title_full_unstemmed A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title_short A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans
title_sort new insect cell line engineered to produce recombinant glycoproteins with cleavable n-glycans
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689212/
https://www.ncbi.nlm.nih.gov/pubmed/34838817
http://dx.doi.org/10.1016/j.jbc.2021.101454
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