FUT8-directed core fucosylation of N-glycans is regulated by the glycan structure and protein environment

García-García, Ana, Serna, Sonia, Yang, Zhang, Delso, Ignacio, Taleb, Víctor, Hicks, Thomas, Artschwager, Raik, Vakhrushev, Sergey Y., Clausen, Henrik, Angulo, Jesús, Corzana, Francisco, Reichardt, Niels C. and Hurtado-Guerrero, Ramon (2021) FUT8-directed core fucosylation of N-glycans is regulated by the glycan structure and protein environment. ACS Catalysis, 11 (15). pp. 9052-9065. ISSN 2155-5435

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Abstract

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Item Type: Article
Additional Information: Acknowledgements: We thank ARAID, Ministerio de Ciencia e Innovación (BFU2016-75633-P and PID2019-105451GB-I00 to R.H.-G. and RTI2018-099592-B-C21 to F.C.), and Gobierno de Aragón (E34_R17 and LMP58_18) with FEDER (2014-2020) funds for “Building Europe from Aragón” for financial support. A.G.-G. thanks Gobierno de Aragón for a predoctoral fellowship. This work was supported by the Lundbeck Foundation, Novo Nordisk Foundation, Innovation Fund Denmark, and the Danish National Research Foundation (DNRF107). J.A. thanks funding from the Biotechnology and Biological Sciences Research Council (BBSRC) through a New Investigator grant and from the Spanish Ministry of Science, Innovation and Universities through the PID2019-109395GB-I00 project. I.D. thanks European Commission for an MSCA-IF-EF-ST grant (PyroSul, ID 890779). T.H. is supported by the UKRI Biotechnology and Biological Sciences Research Council Norwich Research Park Biosciences Doctoral Training Partnership grant BB/M011216/1. N.C.R, S.S., and A.R. acknowledge funding from the Ministry of Science and Education (MINECO) Grant No. CTQ2017-90039-R and RTC-2017-6126-1 and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency-Grant No. MDM-2017-0720. We thank Prof. Thomas Gerken from Case Western Reserve University for proofreading the manuscript.
Uncontrolled Keywords: complex n-glycans,core fucosylation,enzyme kinetics,fut8,high-mannose n-glycans,n-glycosylation,paucimannose-type n-glycans,std nmr,catalysis,chemistry(all) ,/dk/atira/pure/subjectarea/asjc/1500/1503
Faculty \ School: Faculty of Science > School of Pharmacy
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Depositing User: LivePure Connector
Date Deposited: 08 Jun 2022 08:30
Last Modified: 21 Jun 2022 08:36
URI: https://ueaeprints.uea.ac.uk/id/eprint/85460
DOI: 10.1021/acscatal.1c01698

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