PONE-D-24-22138Interplay between de novo and salvage pathways of GDP-fucose synthesisPLOS ONE

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This research was funded by the National Science Centre (Narodowe Centrum Nauki, NCN), Poland, grant number 2022/45/N/NZ3/00093

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The work reported by Edyta Skurska et al., titled "Interplay between de novo and salvage pathways of GDP-fucose synthesis," offers valuable insights into how enzymes are regulated in human knockout cell lines and their significance in fucose metabolism. It provides a comprehensive exploration of the interaction between different pathways involved in GDP-fucose synthesis, shedding light on their roles in cellular processes and potential implications for health and disease.

The authors investigated the interdependency of various enzymes within the two primary pathways of GDP-fucose synthesis. Their study adeptly utilized in vivo assays on human HEK293T cell lines, where they generated knockouts of key enzymes from both the de novo and salvage pathways.

This manuscript primary assertion is that previous studies overlooked the interaction between the de novo and salvage pathways of GDP-fucose synthesis. This study represents the first comprehensive report elucidating the roles of enzymes crucial to GDP-fucose production. The authors successfully demonstrated GDP-fucose formation and observed the response of fucosylated structures in knockout strains when supplemented with free fucose.

Overall, the study illuminates the roles of three key enzymes—GMDS, TSTA, and fucokinase—in knockout strains. Despite blocking the expression of the major salvage pathway enzyme, fucokinase, the levels of GDP-fucose and fucosylated glycans remained unchanged. The experimental findings are robustly supported by the presented data and are effectively communicated in the article.

Minor points :

• In Page 8, line no. 172, The author states that the GDP-fucose concentration in unfed TSTA3KO cells was estimated as 0 µM. What is the inherent cellular concentration of GDP fucose without external supplementation?

• Observed the presence of multiple bands in FCSK knockout cell line (figure 4D)

• "What methods are available to quantify the reaction intermediate GDP-4-keto-6-deoxy mannose in biochemical studies?"

• Is it possible to visually detect the upregulation or downregulation of SLC35C1 in knockout cell lines

Reviewer #2: The manuscript highlights fucose as a critical component of glycoconjugates involved in numerous biological processes in mammalian cells, such as cell adhesion, tissue development, angiogenesis, fertilization, malignancy, and tumor metastasis. The active form of fucose, GDP-fucose, is synthesized through two main pathways: the de novo pathway and the salvage pathway. They describe the enzymes involved in both pathways. The study used the CRISPR/Cas9 system to create cell lines deficient in TSTA3 (TSTA3KO), GMDS (GMDSKO), and FCSK (FCSKKO) in HEK293T cells. Their key finding and novelty is that they highlight a mutual regulation between de novo and salvage pathway enzymes. Results that lead to this finding are:

• Cells lacking TSTA3 produced high amounts of GDP-fucose upon fucose supplementation, unlike GMDS-deficient cells.

• After fucose supplementation, fucokinase levels were elevated in TSTA3-deficient cells, but not in GMDS-deficient cells.

• TSTA3 protein levels increased in FCSK-deficient cells.

• Differences in fucose uptake were observed between the studied cell lines.

The manuscript is acceptable after a few minor changes.

Comments:

Introduction section: The manuscript will significantly benefit by providing more context about the significance of fucose in glycoconjugates and why its study is essential. Consider elaborating on how defects in fucosylation can lead to diseases, which would emphasize the biological importance and relevance of the study.

Figure legend Fig 1B: Line 35: 'graph illustrating the fucose-derived pathway of GDP-fucose synthesis.' Fig 1B is also a linear schematic representation of the Salvage pathway. Why do the authors call it a graph?

Line 45: 'Recently, the second mechanism of fucose delivery, depending on glucose transporter 1 (GLUT1) activity, was described'. I think the authors should use the word 'uptake' instead of delivery to maintain uniformity.

Line 47: 'The depletion of genes encoding enzymes of the de novo GDP-fucose biosynthesis pathway was applied to produce afucosylated antibodies.' This line seems to disturb the flow of the manuscript. Consider beginning the sentence like 'To illustrate the importance of fucosylated antibodies…'

Material and Methods section:

Line 93: 'According to the manufacturer's protocol, HEK293T GMDSKO and FXKO cell lines.' Do the authors mean the FCSKKO cell line? If so, please use FCSKKO throughout the manuscript. Ensure to check Table 1 as well.

Results section:

Line 191: 'GMDS is the second enzyme of the de novo biosynthesis pathway.' GMDS is the first enzyme.

Line 215: 'The addition of fucose in the concentration of 10 μM already caused an increase in GDP-fucose levels in TSTA3KO cells to the rate between ~100 and 600 μM.' Is this statement in the context of Figure 3A? If so, it is unclear which histogram is being talked about. I think you are referring to the third histogram in the graph, which corresponds to 50 uM fucose. Is that right?

The authors mention a number besides the TSTA3KO and GMDSKO lines throughout the figures. It would be helpful to mention the context of these numbers once at the beginning of the results section. Are these different cell lines or just a different replica?

Line 247: 'However, the FCSK protein level was elevated in one of the clones of TSTA3KO cells, regardless of fucose supplementation'. For Fig 4: Please mark an arrow or box in the western blots on the lane you want the readers' attention to.

Why do you observe two bands for FPGT (Fig 4A)?

Figure 5: The figure shows 'HAFUK' and 'cmycFPGT'. Please briefly mention these tags/constructs in the results so it will be easier to follow the western blots.

Figure 7 A and B: Do you have better-looking western blots?

Discussion section:

Feedback Regulation Mechanism:

TSTA3 might be involved in feedback regulation where the levels of GDP-fucose produced via the de novo pathway influence the activity or expression of enzymes in the salvage pathway.

When TSTA3 activity is disrupted, the cellular pool of GDP-fucose might signal an upregulation of salvage pathway enzymes, such as fucokinase (FCSK) and GDP-fucose pyrophosphorylase (FPGT), to compensate for the reduced GDP-fucose synthesis.

If the authors could comment on the above in their discussion, it would enhance the manuscript's readability.

Does TSTA3 form complexes with other enzymes involved in GDP-fucose biosynthesis, stabilizing them and enhancing their activities?

Does TSTA3 influence the transcription of genes encoding enzymes of both pathways?

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Reviewer #1: No

Reviewer #2: No

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

PONE-D-24-22138R1

Dear Dr. Olczak,

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Kind regards,

Ashutosh Pandey, Ph.D.

Academic Editor

PLOS ONE

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