PONE-D-22-34778Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesisPLOS ONE

Dear Dr. Furuichi,

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.

The manuscript is well scheduled and performed. However, based on the Reviewers' comments some issues have arisen. Thus, the reviewers feel that it is well corroborated that SLC35A3 is involved in GAG synthesis but the differential effects of SLC35A3 on the different glycosylation types are lacking. The analysis of the N- and O- glycosylation status of the KO mice chondrocytes KO would strenghten the correlation. This needs to be responnded to and at least disccused. Furthermore how can the authors explain in their HPLC analysis (Supp figure 2) that the SLC35A3 deficiency affects more the peak 3, corresponding to ∆HexUA-GlcN(NS), than the peak 2 corresponding to ∆HexUA- GlcNAc(6S)?? Is it a bias due to the specificities of the enzymes used to cleave the GAGs chains? This needs to be disccussed. Furthermore, please elaborate on the technical querries and minor points raised by the reviewers.

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Additional Editor Comments:

The manuscript is well scheduled and performed. However, based on the Reviewers' comments some issues have arisen. Thus, the reviewers feel that it is well corrobortaed that SLC35A3 is involved in GAG synthesis but the differential effects of SLC35A3 on the different glycosylation types are lacking. The analysis of the N- and O- glycosylation status of the KO mice chondrocytes KO mice would strenghten the correlation. This needs to be responnded to and at least disccused. Furthermore how can the authors explain in their HPLC analysis (Supp figure 2) that the SLC35A3 deficiency affects more the peak 3 corresponding to ∆HexUA-GlcN(NS) than the peak 2 corresponding to ∆HexUA- GlcNAc(6S)?? Is it a bias due to the specificities of the enzymes used to cleave the GAGs chains? This needs to be disccussed. Furthermore, please elaborate on the technical querries and minor points raised by the reviewers.

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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: In this paper Saito et al generated a Slc35a3 knock-out mouse colony using the CRISPR/Cas9 genome editing system with the objective of elucidating the role of SLC35A3 in normal condition and in SLC35A3 related diseases.

Mutant mice were perinatal lethal with a severe chondrodysplasia phenotype including vertebral and growth plate anomalies caused by decreased GAG synthesis demonstrated by GAG disaccharide analysis.

The experiments are described in sufficient details and the approaches used to support the results are up to date.

MAJOR COMMENTS:

In the results, GAG synthesis in cartilage from mutant and wt mice is measured by quantitation of disaccharides from different GAG species in nmol/mg total protein. However in the results and also in the Material and Methods session it is not specified at which step of the experiment total proteins have been measured and the assay which has been used.

I’d suggest to mention that the RC, FC and HC zones in Fig 4 and 5 point to resting, proliferative and hypertrophic zones of the growth plate

MINOR COMMENTS

There are some inaccuracies that should be checked:

Page 8. Line 4 from bottom “lumber vertebrae”

Page 12 line 10 from top “embryos et E18.5” and line 15 from top “lumber vertebrae”

Page 14 line 16 from top “Sic35a3-/-“

Page 15 lines 6 and 7 from top “lumber vertebrae”

Page 18 line 11 from top “Slc35d-/-“

Reviewer #2: The paper of Soichiro Saito and collaborators investigated the relationship between SLC35A3 and GAG synthesis by generating a KO SLC35A3 mice. They nicely showed that mice exhibited lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis. This is an interesting paper bringing more evidences that SLC35A3 is involved in GAG synthesis. The first part of the paper is very strong and well done with the generation and the KO SLC35A3 and its phenotyping. The second part on the GAG analysis is more difficult to follow and constitutes the weakest part of the paper. I understand the difficulty of these analysis but as it is, the paper stays descriptive. The mouse recapitulates the SLC35A3 human phenotype with major skeletal abnormalities due to GAG defects. This is known that SLC35A3 is involved in GAG synthesis (see my minor point). I also have the feeling that we miss the fundamental study on trying to explain the differential effects of a lack of SLC35A3 on the different glycosylation types. It would have brought a lot if the authors could have analyzed the N- and O- glycosylation status of the chondrocytes KO mice. This is my major criticism.

Major point:

Coming back to the GAGs analysis by HPLC after enzymatic digestions, how can the authors explain in their analysis (Supp figure 2) that a deficiency affects more the pick 3 corresponding to ∆HexUA-GlcN(NS) than the pick 2 corresponding to ∆HexUA- GlcNAc(6S)?? Is it a bias due to the specificities of the enzymes used to cleave the GAGs chains? This can be annoying.

I fully agree with a general GAGs defect from the experiments but the differential quantification defect on HS/ KS/ DS is much more difficult to assess from the experiments. This “strong” GalNAc defect is also interesting but stays descriptive. This is where O-GalNAc glycosylation analysis would be very interesting in confirming this defect.

Minor point:

The authors say that “the relationship between the function of SLC35A3 and GAG biosynthesis is largely unknown”. I do not agree with this sentence. The link has been evidenced in Haouari’s paper on serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies. The clear defect observed on bikunin in SLC35A3 patient strongly demonstrates that GAGs are affected.

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Reviewer #1: No

Reviewer #2: No

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Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis

PONE-D-22-34778R1

Dear Dr. Furuichi,

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.

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Kind regards,

Dragana Nikitovic, 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 #2: All comments have been addressed

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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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: 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: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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