PONE-D-19-20746

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PLOS ONE

Dear Dr. Venugopal,

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 Oct 02 2019 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Dr. Sakamuri V. Reddy

Academic Editor

PLOS ONE

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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: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: Major Comments:

1) In Results Section the Figures are not cited sequentially. It is cited like Fig. 2, 3 then 6A, 6B, Fig 3, then Fig. 7, Fig.4, Fig 5 and Fig. 8. Authors need to completely revise the Results section and explain the results according to the sequence of the figures or change the Figure numbers.

2) Figure legends are not written properly and completely. For example, in many Figure legends the results are explained and then experimental detail. There is no need to write the results in Figure legends. Please write the experimental details and number of repeat experiments.

3) Figures 6a and 6b should be numbered as Fig. 6 and Fig. 7 respectively.

3) It is not clear that why all mutants TRACPs were detected intracellularly to a similar level? Please discuss.

3) Discussion is lengthy. It should be shortened.

Minor comments:

There are some typo and grammatical errors, that need to be corrected.

Reviewer #2: PONE-D-19-20746

Characterization of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatases Associated with Spondyloenchondrodysplasia

This paper characterizes the Acp5 mutations encoding TRACP that commonly associated with Spondyloenchondrodysplasia (SPENCD). SPENCD is a recently identified disease, and it seems promising and exciting to investigate such disorder at the molecular level.

However, while reading the MS, I felt lost. Data is impressive. Clearer presentation is needed. The overall idea was not well-explained. MS needs rewriting. Some of the general and specific comments are stated below:

• General comments:

o The overall purpose of this study was not stated clearly.

o Excessive explanation in the result section. This makes some sorts of confusion. Every result subsection should plainly and simply state the result, whereas any other explanations should be carried out to the "discussion” section.

o There should be more introductions about Spondyloenchondrodysplasia and TRACP proteins in general. The relationship between the diseases and TRACP has to be elaborated in much detail.

o This reviewer is not clear about the bands in the Western analyses. MW markers are not provided. Several lanes are provided with no lane numbers.

o How do you normalize the secreted and intracellular proteins? Why do you think the top band as a non-specific band and consider that as a loading control? For secreted proteins in media, it is essential to show a Coomassie blue stained gel as a loading control. GAPDH or any other intracellular loading control should be added for normalization of the intracellular proteins

• Specific comments:

o The underlying reason for using HEK-293 cell line is not stated at all. Why not using other cell lines that not ubiquitously expressing TRACP?

o Figure 2: No clear illustration or indication of the stainable activity of cells. Quantification would also help.

o Result: the result section should correspond with the numerical order of the figures. The readers must not jump between figures while reading.

o Results: figure 6B does not correlate with what mentioned in the corresponding result section.

o Figures: Western analyses: comments provided under general comments

o Figures: Quantification alone for figure 7 is not sufficient. It is better to provide visual representations such as immunostaining and Western blotting analyses.

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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.

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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.

PONE-D-19-20746R1

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PLOS ONE

Dear Dr. Venugopal,

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.

ACADEMIC EDITOR: I suggest the authors to proof read the manuscript for English language and follow the journal format as noted in the reviewers comments below. Please address the  several minor comments raised by rev#3 carefully.

We would appreciate receiving your revised manuscript by Jan 18 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,

Dr. Sakamuri V. Reddy

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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 #1: No

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 #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

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 #1: There is only slight improvement in the revised manuscript.

Major issues:

1) Authors have not provided point-by-point response in detail. Very casual approach is taken by authors in writing of the response to reviewers comments.

2) There is only slight improvement in English writing in the manuscript.

3) However, the scientific language is still very poor throughout the manuscript, particularly the authors have not used proper scientific terms and framing of the sentences and grammar. It appears that manuscript is not properly edited by senior authors.

3) Result section is still very poor. Results of all the experiments are not written in details. The sub-figures are not cited in the text. Although, there are several sub-figures in many figures such as Figures 3, 4, 5 and 9, authors have just written there numbers.

4) Many Figures Legends are incomplete. e.g. In Figure 1 Legend, it is mentioned that missence mutations are shown below the WT sequence. This is not reflected in actual figure. Similarly, there are many mistakes in other Figures legends.

Reviewer #2: PONE-D-19-20746R1

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid

Phosphatase Associated with Spondyloenchondrodysplasia

The MS has been revised as suggested, and the authors corrected the crucial issues.

It can be accepted for publication

Reviewer #3: Comments to the Author:

This article attempt to characterize the eight already reported missense mutations in Acp5 which is associated with spondyloenchondrodysplasia (SPENCD). The authors identified that the presence of mutants TRACP proteins are unable to induce enzymatic activity. Further, except T89I and M264K mutant proteins, others are in denatured precursor forms. The authors concluded that understanding the structural and functional aspects of mutant TRACP could lead to better understating of immune response and bone metabolism as well as targeting drug development.

The concept of the paper is relevant however there are many concerns that should be addressed before publication. The authors should follow the scientific writing format rather than common words and sentences. The whole manuscript should be corrected for details, clarity, and grammar.

Major issues:

1) In Abstract: RT-qPCR analysis is mentioned but there is no such figure and result in appropriate sections.

2) Methods section written poorly, refer the articles and follow the formats. For instance, western blotting is written without clarity and details. Some of the Methods written like results that should be avoided.

3) It is not appropriate to write a sentence like this in the result section “Other salient features of TRACP are described in Figure 1 legend”. Provide more details about TRACP enzyme in this section.

4) “The steady state transcription ….10-fold lower compared to WT”. There is no corresponding figures/results in this manuscript.

Minor issues:

1) In Abstract: At the end, summarize or conclude the work and it should exactly reflect the results. Then write what it could be useful to, such as drug development and underlying mechanisms, etc.

2) In Abstract: “Certainly, determining the structure functional relationships in TRACP will expand….” Should modify to “Determining the structural and functional relationships of TRACP may expand… “

3) An unscientific sentence like this and many others throughout the manuscript should be modified “TRACP has many functions in vitro and its biological role in bone resorption and immune responses is becoming clearer”. What exactly authors want to convey with this sentence “TRACP has many functions in vitro….”

4) “Therefore, serum TRACP5a may serve as a marker for systemic macrophage number…” is it possible to count the macrophage number using serum TRACP5a level? Similarly, isoform 5b release and osteoclast number? Clarify both the statements.

5) Mentioned in this sentence that “We and others have shown earlier [9, 10] that mutations…” but there is one other’s paper cited, add more reference or modify.

6) Follow the same format for gene name, ACP5 or Acp5.

7) Add references to these sentences and many others "More specifically, ….TRACP enzyme”, “Other salient features…..calcification”

8) “Transfection of synthetic genes” This subtitle should be written like this “Transformation of mutant genes and subcloning” and describe the methods in the text.

9) “Transient transfection and generation of stable cell line” This should be a subtitle for the next paragraph (from this sentence onwards “HEK-293 cells were transfected with……recommended procedures.”

10) What is the loading control used for western blot analysis? Explain.

11) Figures should be labeled appropriately. For instance, Fig 2, TRAP staining and Mab220.

12) Y-axis scale difference is high between min and max in many bar graphs, Therefore if it appropriate to represent the graph with the broken y-axis to show the lower values.

13) The results section should be written clearly with details.

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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 #1: No

Reviewer #2: No

Reviewer #3: 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.

Characterisation of Acp5 Missense Mutations Encoding Tartrate-Resistant Acid Phosphatase Associated with Spondyloenchondrodysplasia

PONE-D-19-20746R2

Dear Dr. Venugopal,

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,

Sakamuri V. Reddy, 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 #1: 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 #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: 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 #1: 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 #1: 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 #1: Sub figures such as A, B, C etc.of Figures 4, 5 are not yet cited in Resullts. That need to be corrected.

Reviewer #3: (No Response)

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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 #1: No

Reviewer #3: No