Human and mouse activin genes: Divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B

Paul C. Billings; Candice Bizzaro; Evan Yang; Juliet Chung; Christina Mundy; Maurizio Pacifici

doi:10.1371/journal.pone.0229254

Peer Review History

Original SubmissionJanuary 7, 2020
30 Jan 2020 Decision Letter - Xin Zhang, Editor PONE-D-20-00545 Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B PLOS ONE Dear Dr. Billings, 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. We would appreciate receiving your revised manuscript by Mar 15 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Xin Zhang, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: 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 ****** 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: No Reviewer #2: Yes ****** 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: This MS entitled: Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B” by Billings et al reports the gene expression of Activins in mouse. It is a related work to the previous published paper (Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function published in the same journal 2019). The data are clear and the paper is well-written. Reviewer #2: In this study, the authors investigated the differential expression of Activin protein variants in humans and mice, and analyzed their interaction with HS. They reported the absence of HBD in mouse Act A, which is instead identified in mouse and human Act B. These findings imply a novel and potentially important mechanism that regulates the distribution and function of activin (hetero-)dimers within the extracellular milieu. Overall the manuscript is well written and the data are appropriately presented. I have only the following points that the authors can consider commenting on or discussing in more detail. Major point 1. In Act B, HBD is located in the pro-region, not the mature ligand. Considering the localization of HS chains on cell surface, it is important for the authors to clarify whether the dimers are secreted as full-length protein with mature ligand still associated with the pro-region after intracellular cleavage by furin/proprotein convertases. Or, whether the pro-regions interact with the ECM alone or with the mature growth factor after secretion. Minor points 1. [Abstract] “Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature N-terminal ligand” and [Introduction] “resulting in a long C-terminal pro-region and a N-terminal active mature ligand”: should be N-terminal pro-region and a C-terminal mature ligand. 2. Fig. 4: Annotation for the colors of the protein structures is desired. 3. Typo: P16, L22: “sold” should be “solid”. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0229254.r001
Revision 1
31 Jan 2020 Author Response January 31, 2020 RE: PONE-D-20-00545- Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B Xin Zhang, Ph.D. Academic Editor PLOS ONE Dear Dr. Zhang: Thank you for your prompt review of our manuscript. We have enclosed our revised manuscript and have addressed the comments of each Reviewer and Editor below. Corrections and changes in the revised manuscript are in bold type. Reviewer #1: This MS entitled ‘Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B’ by Billings et al reports the gene expression of Activins in mouse. It is a related work to the previous published paper (Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function published in the same journal 2019). The data are clear and the paper is well-written. Response We thank Reviewer #1 for their constructive evaluation of our work. Reviewer #2: In this study, the authors investigated the differential expression of Activin protein variants in humans and mice, and analyzed their interaction with HS. They reported the absence of HBD in mouse Act A, which is instead identified in mouse and human Act B. These findings imply a novel and potentially important mechanism that regulates the distribution and function of activin (hetero-) dimers within the extracellular milieu. Overall the manuscript is well written and the data are appropriately presented. I have only the following points that the authors can consider commenting on or discussing in more detail. Major point 1. In Act B, HBD is located in the pro-region, not the mature ligand. Considering the localization of HS chains on cell surface, it is important for the authors to clarify whether the dimers are secreted as full-length protein with mature ligand still associated with the pro-region after intracellular cleavage by furin/proprotein convertases. Or, whether the pro-regions interact with the ECM alone or with the mature growth factor after secretion. Minor points 1. [Abstract] “Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature N-terminal ligand” and [Introduction] “resulting in a long C-terminal pro-region and a N-terminal active mature ligand”: should be N-terminal pro-region and a C-terminal mature ligand. 2. Fig. 4: Annotation for the colors of the protein structures is desired. 3. Typo: P16, L22: “sold” should be “solid”. Response We thank Reviewer #2 for their careful and constructive evaluation of our work. Major point 1. As requested by the Reviewer, we have addressed the issue of Activin secretion and have added the following statement to the Discussion section. It is generally assumed that activins are synthesized, dimerize and undergo proteolytic cleavage by furin/proprotein convertases intracellularly and are subsequently secreted as complexes containing the N-terminal pro-region non-covalently associated with the C-terminal mature ligand. Following secretion, this complex binds HSPGs or other ECM components and the mature ligand is released by an unknown mechanism [17]. Minor points 1. As suggested by the reviewer, we have revised the statement in the Abstract to read “Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C-terminal ligand”. We have corrected the statement in the Introduction to read “resulting in a long N-terminal pro-region and a C-terminal mature ligand. 2. Fig 4, legend. As requested, we have indicated the colors of the protein structures as follows: The large and highly structured N-terminal pro-region (yellow) continues with the C-terminal mature ligand (green). The signal peptide on the N-terminus is in red while the Furin cleavage site is at the arrow. 3. Typo: P16, L22: As requested, “sold” has been corrected to “solid”. Editorial Comments 1. As requested by the Editor, we have included uncropped images of gels in Supporting information- S5 Fig. Uncropped western blots shown in Fig 7 of the manuscript. 2. As requested, the section on use of animal tissue has been revised and the following statement has been added to the Methods section. Tissue Isolation from Mice All studies were approved by the Institutional Care and Use Committee (IACUC), Children’s Hospital of Philadelphia under Protocol # IAC 17-000958. We would like to thank the Reviewers and Editorial staff for their prompt and constructive evaluation of our work. We hope that our revised manuscript will be acceptable for publication. Sincerely, Paul Billings, Ph. D. Division of Orthopaedic Surgery The Children's Hospital of Philadelphia Philadelphia, PA 19104 Ph: 267-426-9691/425-2077 Email: BillingsP@email.chop.edu Attachments Attachment Submitted filename: Rebuttal-Act manu 2_1.31.20.docx https://doi.org/10.1371/journal.pone.0229254.r002
4 Feb 2020 Decision Letter - Xin Zhang, Editor Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B PONE-D-20-00545R1 Dear Dr. Billings, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Xin Zhang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0229254.r003
Formally Accepted
11 Feb 2020 Acceptance Letter - Xin Zhang, Editor PONE-D-20-00545R1 Human and mouse activin genes: divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B Dear Dr. Billings: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Xin Zhang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0229254.r004

Open letter on the publication of peer review reports

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. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .