Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response

Yuki M. Kyunai; Mika Sakamoto; Mayuko Koreishi; Yoshio Tsujino; Ayano Satoh

doi:10.1371/journal.pone.0281516

Peer Review History

Original SubmissionSeptember 22, 2022
22 Sep 2022 Author Response Attachments Attachment Submitted filename: rebut_ed.docx https://doi.org/10.1371/journal.pone.0281516.r001
11 Jan 2023 Decision Letter - Jian Xu, Editor PONE-D-22-26220Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response.PLOS ONE Dear Dr. Satoh, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Feb 25 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors addressed the reviewers' concern properly. The reviewer encourages does not have any further comments. The reviewer expects that the authors reveal the mechanistic insights why the FUT8 expression is upregulated during oxidative stress conditions in the next work. Reviewer #2: In the present manuscript, authors reported the relationship between changes in glycosylation and oxidative stress by using GlycoMaple. Unfortunately, this reviewer believes that the present data do not support the conclusions. The changes in core fucosylation should be confirmed by mass spectrometry, since the UEA-I lectin used in the present study is not specific for core fucosylation. Therefore, the present version of the study is not suitable for publication in this journal. Reviewer #3: The authors demonstrated that the treatment with 5H4PB and SFN upregulated FUT8 gene and induced its product, core fucose structure. This is a good example to examine the glycan structure by using the in silico glycan structure prediction tool GlycoMaple. However, the current version is too preliminary for the publication. � This study lacks positive control experiments for oxidative stress responses. The authors should reanalyze with H2O2 to conclude the upregulation of the FUT8 gene by the oxidative stress response. � Why did the authors analyze HaCaT cells? The authors may have a reasonable reason for this experiment. � In the flow cytometric analysis in Figure 3, the induction of core fucose structure is significant but very weak. This flow cytometric analysis lacks staining controls. Lectin blotting is another good way to see core fucose structure. LCA or PhoSL would be much better lectin to identify core fucose. � The authors also found a strong downregulation of the MGAT3 gene in Figure 1. How about bisecting GlcNAc structure? The authors should select more suitable lectins for analyzing core fucose. � In the Discussion part, do not repeat the description mentioned in the Result part. Instead of it, the authors should discuss the relationship between glycan and oxidative stress response more deeply. � The authors mentioned that glycan changes by oxidative stress is the first report but it is not true. The following papers have been already published. 1) Khoder-Agha F, Kietzmann T. The glyco-redox interplay: Principles and consequences on the role of reactive oxygen species during protein glycosylation. Redox Biol. 2021 Jun;42:101888. doi: 10.1016/j.redox.2021.101888. Epub 2021 Feb 10. PMID: 33602616; PMCID: PMC8113034. 2) Taniguchi N, Kizuka Y, Takamatsu S, Miyoshi E, Gao C, Suzuki K, Kitazume S, Ohtsubo K. Glyco-redox, a link between oxidative stress and changes of glycans: Lessons from research on glutathione, reactive oxygen and nitrogen species to glycobiology. Arch Biochem Biophys. 2016 Apr 1;595:72-80. doi: 10.1016/j.abb.2015.11.024. PMID: 27095220. 3) Taniguchi N, Takahashi M, Kizuka Y, Kitazume S, Shuvaev VV, Ookawara T, Furuta A. Glycation vs. glycosylation: a tale of two different chemistries and biology in Alzheimer's disease. Glycoconj J. 2016 Aug;33(4):487-97. doi: 10.1007/s10719-016-9690-2. Epub 2016 Jun 21. PMID: 27325408. 4) Lim SY, Ng BH, Vermulapalli D, Lau H, Carrasco Laserna AK, Yang X, Tan SH, Chan MY, Li SFY. Simultaneous Polar Metabolite and N-Glycan Extraction Workflow for Joint-Omics Analysis: A Synergistic Approach for Novel Insights into Diseases. J Proteome Res. 2022 Mar 4;21(3):643-653. doi: 10.1021/acs.jproteome.1c00676. Epub 2022 Jan 24. PMID: 35073107. � ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0281516.r002
Revision 1
19 Jan 2023 Author Response Reviewer #1: The authors addressed the reviewers' concern properly. The reviewer encourages does not have any further comments. The reviewer expects that the authors reveal the mechanistic insights why the FUT8 expression is upregulated during oxidative stress conditions in the next work. We appreciate your constructive comments and suggestions that have improved our manuscript. Reviewer #2: In the present manuscript, authors reported the relationship between changes in glycosylation and oxidative stress by using GlycoMaple. Unfortunately, this reviewer believes that the present data do not support the conclusions. The changes in core fucosylation should be confirmed by mass spectrometry, since the UEA-I lectin used in the present study is not specific for core fucosylation. Therefore, the present version of the study is not suitable for publication in this journal. Since we agree with you that lectin UEA-I does not label only the core fucose, we had already added the following description at the end of the Results section in the original version of our manuscript: The cell line used in this study expressed only Fut 1, 2, 8, 10, and 11 under the conditions tested, and only Fut 8 showed an increase with the drug treatments. Since fucosyltransferases other than Fut 8 were not expressed or were not increased with the drug treatment, we concluded that the increase in reactivity to UEA-I was a result of the increase in Fut 8 expression. Reviewer #3: The authors demonstrated that the treatment with 5H4PB and SFN upregulated FUT8 gene and induced its product, core fucose structure. This is a good example to examine the glycan structure by using the in silico glycan structure prediction tool GlycoMaple. However, the current version is too preliminary for the publication. � This study lacks positive control experiments for oxidative stress responses. The authors should reanalyze with H2O2 to conclude the upregulation of the FUT8 gene by the oxidative stress response. We found two datasets (NCBI BioProject: PRJNA200279, PRJNA252456) that included HaCaT cells exposed to H2O2 and analyzed them. We are reluctant to include these results in the manuscript because they are not consistent with the main objective of our study, which was to investigate the induction of the antioxidant response. Specifically, the expression of phase II antioxidant enzymes, the key signature of the antioxidant response, including NQO1 and GCLC, did not show an increase in cells treated with H2O2, suggesting that H2O2 treatment may not induce the desired response. � Why did the authors analyze HaCaT cells? The authors may have a reasonable reason for this experiment. As stated in the Introduction section of the original manuscript, it is believed that the antioxidant response in the skin, particularly in keratinocytes, which is the source of the HaCaT cell line used in this study, is associated with skin sensitization, allergy, and dermatitis. We believed that identifying a marker that could detect early symptoms of such conditions could contribute to the diagnostic process. � In the flow cytometric analysis in Figure 3, the induction of core fucose structure is significant but very weak. This flow cytometric analysis lacks staining controls. Lectin blotting is another good way to see core fucose structure. LCA or PhoSL would be much better lectin to identify core fucose. Following your suggestion, we performed Lectin UEA-I blotting as shown below. The drug treatment increased the UEA-I signal approximately 1.3-fold. The signal was normalized by anti-tubulin Western blotting. Lanes 1 and 2 show control and drug treated, respectively (left panel). We also performed FACS analysis using LCA as shown in the right panel. After the drug treatment, the LCA signal increased 2.8-fold, confirming the increased core fucose structure by the drug treatment. � The authors also found a strong downregulation of the MGAT3 gene in Figure 1. How about bisecting GlcNAc structure? The halving of bisecting GlcNAc detected by lectin WGA (shown below) is consistent with the decrease in MGAT3 expression caused by the drug treatment (Figure 1), as expected. However, the focus of the current study is on Fut8, so this point is not included in this manuscript. The authors should select more suitable lectins for analyzing core fucose. We agree with you that the lectin UEA-I may not be ideal for core fucose labeling. However, we believe that our use of UEA-I is appropriate for the reasons described at the end of the Results section of the original version of our manuscript: The cell line used in this study expressed only Fut 1, 2, 8, 10, and 11 under the conditions tested, and only Fut 8 showed an increase with the drug treatments. Since fucosyltransferases other than Fut 8 were not expressed or were not increased with drug treatment, we concluded that the increase in reactivity to UEA-I was a result of the increase in Fut 8 expression. � In the Discussion part, do not repeat the description mentioned in the Result part. Instead of it, the authors should discuss the relationship between glycan and oxidative stress response more deeply. � The authors mentioned that glycan changes by oxidative stress is the first report but it is not true. The following papers have been already published. We sincerely appreciate your bringing these references to our attention. We have now included them in the revised manuscript and also added and reorganized the discussion based on them. Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We have made the necessary adjustments to the best of our ability. Please let us know if any further corrections are needed. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. It’s available at https://www.ncbi.nlm.nih.gov/bioproject/897738 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. We decided not to include the related information, as it was deemed unnecessary for the understanding of the manuscript. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0281516.r003
25 Jan 2023 Decision Letter - Jian Xu, Editor Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response. PONE-D-22-26220R1 Dear Dr. Satoh, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jian Xu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Partly Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: The present study is interesting and OK now. This reviewer hope that the glycan structures need to be determined by MS-analysis, not only lectin. Reviewer #3: This paper was well revised by responding to the reviewers' comments especially involvement of glycan chages by oxidative stress. This paper is now suitable for publication. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No Reviewer #3: Yes ******** https://doi.org/10.1371/journal.pone.0281516.r004
Formally Accepted
31 Jan 2023 Acceptance Letter - Jian Xu, Editor PONE-D-22-26220R1 Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response Dear Dr. Satoh: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jian Xu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281516.r005

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