Applying Acylated Fucose Analogues to Metabolic Glycoengineering
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 biosynthe...
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| Format: | Article |
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MDPI AG
2015-11-01
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| Series: | Bioengineering |
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| Online Access: | http://www.mdpi.com/2306-5354/2/4/213 |
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| author | Julia Rosenlöcher Verena Böhrsch Michael Sacharjat Véronique Blanchard Christoph Giese Volker Sandig Christian P. R. Hackenberger Stephan Hinderlich |
| author_facet | Julia Rosenlöcher Verena Böhrsch Michael Sacharjat Véronique Blanchard Christoph Giese Volker Sandig Christian P. R. Hackenberger Stephan Hinderlich |
| author_sort | Julia Rosenlöcher |
| collection | DOAJ |
| description | 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. |
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| issn | 2306-5354 |
| language | English |
| last_indexed | 2024-03-12T20:24:43Z |
| publishDate | 2015-11-01 |
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| series | Bioengineering |
| spelling | doaj.art-b876e37c4dee42e2967b6abfacb50ab42023-08-02T00:35:20ZengMDPI AGBioengineering2306-53542015-11-012421323410.3390/bioengineering2040213bioengineering2040213Applying Acylated Fucose Analogues to Metabolic GlycoengineeringJulia Rosenlöcher0Verena Böhrsch1Michael Sacharjat2Véronique Blanchard3Christoph Giese4Volker Sandig5Christian P. R. Hackenberger6Stephan Hinderlich7Laboratory of Biochemistry, Department of Life Sciences and Technology, Beuth University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin, GermanyLeibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, GermanyProBioGen AG, Goethestrasse 54, 13086 Berlin, GermanyInstitute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyProBioGen AG, Goethestrasse 54, 13086 Berlin, GermanyProBioGen AG, Goethestrasse 54, 13086 Berlin, GermanyLeibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, GermanyLaboratory of Biochemistry, Department of Life Sciences and Technology, Beuth University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin, GermanyManipulations 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.http://www.mdpi.com/2306-5354/2/4/213acylationfucose analoguesfucosylationglycosylationmetabolic glycoengineeringsalvage pathway |
| spellingShingle | Julia Rosenlöcher Verena Böhrsch Michael Sacharjat Véronique Blanchard Christoph Giese Volker Sandig Christian P. R. Hackenberger Stephan Hinderlich Applying Acylated Fucose Analogues to Metabolic Glycoengineering Bioengineering acylation fucose analogues fucosylation glycosylation metabolic glycoengineering salvage pathway |
| title | Applying Acylated Fucose Analogues to Metabolic Glycoengineering |
| title_full | Applying Acylated Fucose Analogues to Metabolic Glycoengineering |
| title_fullStr | Applying Acylated Fucose Analogues to Metabolic Glycoengineering |
| title_full_unstemmed | Applying Acylated Fucose Analogues to Metabolic Glycoengineering |
| title_short | Applying Acylated Fucose Analogues to Metabolic Glycoengineering |
| title_sort | applying acylated fucose analogues to metabolic glycoengineering |
| topic | acylation fucose analogues fucosylation glycosylation metabolic glycoengineering salvage pathway |
| url | http://www.mdpi.com/2306-5354/2/4/213 |
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