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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...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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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. |
format | Online Article Text |
id | pubmed-8689212 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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