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Bioengineering 2015, 2(4), 213-234; doi:10.3390/bioengineering2040213

Applying Acylated Fucose Analogues to Metabolic Glycoengineering

1
Laboratory of Biochemistry, Department of Life Sciences and Technology, Beuth University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin, Germany
2
Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
3
Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany
4
ProBioGen AG, Goethestrasse 54, 13086 Berlin, Germany
5
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
6
Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany
*
Author to whom correspondence should be addressed.
Academic Editors: Mark Blenner and Michael D. Lynch
Received: 30 September 2015 / Revised: 13 November 2015 / Accepted: 23 November 2015 / Published: 30 November 2015
(This article belongs to the Special Issue Metabolic Engineering)
View Full-Text   |   Download PDF [2081 KB, uploaded 30 November 2015]   |  

Abstract

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody’s binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering. View Full-Text
Keywords: acylation; fucose analogues; fucosylation; glycosylation; metabolic glycoengineering; salvage pathway acylation; fucose analogues; fucosylation; glycosylation; metabolic glycoengineering; salvage pathway
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MDPI and ACS Style

Rosenlöcher, J.; Böhrsch, V.; Sacharjat, M.; Blanchard, V.; Giese, C.; Sandig, V.; Hackenberger, C.P.R.; Hinderlich, S. Applying Acylated Fucose Analogues to Metabolic Glycoengineering. Bioengineering 2015, 2, 213-234.

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