Interplay between de novo and salvage pathways of GDP-fucose synthesis

PLOS ONE RESEARCH ARTICLE Interplay between de novo and salvage pathways of GDP-fucose synthesis Edyta Skurska, Mariusz Olczak ID* Department of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Skurska E, Olczak M (2024) Interplay between de novo and salvage pathways of GDPfucose synthesis. PLoS ONE 19(10): e0309450. https://doi.org/10.1371/journal.pone.0309450 Editor: Ashutosh Pandey, Baylor College of Medicine, UNITED STATES OF AMERICA Abstract GDP-fucose is synthesised via two pathways: de novo and salvage. The first uses GDPmannose as a substrate, and the second uses free fucose. To date, these pathways have been considered to work separately and not to have an influence on each other. We report the mutual response of the de novo and salvage pathways to the lack of enzymes from a particular route of GDP-fucose synthesis. We detected different efficiencies of GDP-fucose and fucosylated structure synthesis after a single inactivation of enzymes of the de novo pathway. Our study demonstrated the unequal influence of the salvage enzymes on the production of GDP-fucose by enzymes of the de novo biosynthesis pathway. Simultaneously, we detected an elevated level of one of the enzymes of the de novo pathway in the cell line lacking the enzyme of the salvage biosynthesis pathway. Additionally, we identified dissimilarities in fucose uptake between cells lacking TSTA3 and GMDS proteins. Received: June 5, 2024 Accepted: August 12, 2024 Published: October 24, 2024 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0309450 Copyright: © 2024 Skurska, Olczak. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: 1. National Science Centre (Narodowe Centrum Nauki, NCN), Poland, grant number 2022/ 45/N/NZ3/00093 (to ES) and 2. National Science Introduction Fucose, a crucial component of many glycoconjugate structures, plays a pivotal role in numerous biological processes in mammalian cells. These fucosylated structures are involved in crucial functions such as cell adhesion, tissue development, angiogenesis, fertilisation, malignancy, and tumour metastasis [1–3]. Increasingly, up/downregulation of fucosylation is found in cancer cells [4–6]. Some of them can cause tumour multidrug resistance (MDR) [7, 8]. Besides, it was shown that core fucosylation induced epithelial-mesenchymal transition (EMT) in lung cancer cells, promoting cell migration [9]. Fucosylation also takes part in the development of immune cells [10]. Abnormalities in terminal fucosylation were pointed out as a hallmark of inflammatory macrophages in rheumatoid arthritis [11]. Only its active form, GDP-fucose, is used to synthesise oligosaccharides. In mammals, GDP-fucose is produced via two separately working biosynthesis mechanisms, de novo and salvage pathways [1]. The de novo pathway converts GDP-mannose to GDP-fucose in a threestep enzymatic reaction. Firstly, GDP-mannose 4,6-dehydratase (GMDS) converts GDP-mannose to GDP-4-keto-6-deoxymannose [12], and then GDP-keto-6-deoxymannose-3,5-epimerase (TSTA3), an enzyme of dual activity of epimerase/reductase, transforms it to GDP-fucose [13]. In the salvage pathway, L-β-fucose is phosphorylated by fucokinase (FCSK) [14], and fucose-1-phosphate is converted by GDP-fucose pyrophosphorylase (FPGT) to GDP-fucose PLOS ONE | https://doi.org/10.1371/journal.pone.0309450 October 24, 2024 1 / 21 PLOS ONE Interplay between pathways of GDP-fucose synthesis Centre (Narodowe Centrum Nauki, NCN), Poland, grant number 2023/51/B/NZ3/00810 (to MO) (the second, new source of financing) We declare that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript in both grants. Competing interests: The authors have declared that no competing interests exist. Fig 1. Graphical presentation of GDP-fucose biosynthesis pathways in mammalian cells. (A) scheme presenting the de novo biosynthesis pathway of GDP-fucose. (B) diagram illustrating the fucose-derived pathway of GDP-fucose synthesis. https://doi.org/10.1371/journal.pone.0309450.g001 (Fig 1) [15]. Most GDP-fucose is produced via the de novo pathway, while free L-fucose is reused for the salvage pathway [16, 17]. Free L-fucose is a substrate utilised by fucokinase. It could originate from an extracellular space or lysosomal degradation of fucosylated glycans [14]. Alpha-L-fucosidase (FUCA1) hydrolyses the alpha-linked fucose joined to the N-acetylglucosamine or galactose moieties of glycoproteins in lysosomes [18, 19]. The reaction liberates the α-anomer of L-fucose, which cannot be utilised by fucokinase, as this enzyme uses only the β-version of L-fucose [20]. Fucose mutarotase converts fucose from the α to the β anomer [21]. L-fucose, in its β-anomer, can be obtained from outside the cell. There are at least two mechanisms of uptake of L-fucose by mammalian cells [22]; one is based on macropinocytosis, which is initiated by the sensing of extracellular calcium by the G protein-coupled receptor, calcium-sensing receptor, CaSR [23]. Recently, the second mechanism of fucose uptake, depending on glucose transporter 1 (GLUT1) activity, was described [22]. To illustrate the importance of the fucosylation of antibodies, the depletion of genes encoding enzymes of the de novo GDP-fucose biosynthesis pathway was applied to produce afucosylated antibodies. The lack of fucose in antibodies is well known to enhance the antibodydependent cellular cytotoxicity [24–28]. These studies have shown a complete loss of fucosylated N-glycans decorating synthesised antibodies and indicated that fucosylation of antibodies produced by cell lines deficient in TSTA3 or GMDS can be regulated equally by fucose supplementation in various concentrations. Moreover, there is no information on whether the lack of enzymes taking part in the mannose-derived GDP-fucose biosynthesis pathway affects salvage biosynthesis proteins. In this study, we employed the CRISPR/Cas9 system to select cell lines deficient in TSTA3 (TSTA3KO), GMDS (GMDSKO) and FCSK (FCSKKO) in the human embryonic kidney 293T (HEK293T) cell line. We found that cells lacking the TSTA3 enzyme produced enormously high amounts of GDP-fucose upon fucose supplementation, whereas it does not happen in cells deficient in GMDS protein. We revealed the mutual r (...truncated)