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Influence of protein/glycan interaction on site-specific glycan heterogeneity


Losfeld, Marie-Estelle; Scibona, Ernesto; Lin, Chia-Wei; Villiger, Thomas K; Gauss, Robert; Morbidelli, Massimo; Aebi, Markus (2017). Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB Journal, 31(10):4623-4635.

Abstract

To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity.

Abstract

To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:October 2017
Deposited On:26 Jan 2018 12:28
Last Modified:19 Feb 2018 10:35
Publisher:Federation of American Society of Experimental Biology
ISSN:0892-6638
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1096/fj.201700403R
PubMed ID:28679530

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