PONE-D-20-32839

Altered immunoglobulin G glycosylation in patients with isolated hyperprolactinaemia

PLOS ONE

Dear Dr. Landberg,

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Reviewers' comments:

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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

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

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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: The authors investigated the IgG glycosylation changes in patients with hyperprolactinaemia. Moderate hyperprolactinaemia was found to be associated with increased galactosylation of IgG1, IgG2 and IgG3 though it wasn't confirmed in case of severe hyperprolactinaemia. Level of prolactin may have an impact on IgG interactions with Fc-receptors, complement and lectins, and consequently, lead to an altered immune response. The article is well written and structured, but we have some suggestions which could improve the quality of this manuscript.

Introduction: Macroprolactinaemia, which is mentioned in the text, should be also explained next to hyperprolactinaemia (line 73).

Lines 90-100: The description of different patient groups (including macroprolactinaemia group) is somewhat confusing at times, it would be better to include these data in the Table 1.

Line 192-193: Only fucosylated galactosylated glycans were taken into the equation to calculate galactosylation levels, but the IgG contains also non fucosylated species in lower amounts, as correctly commented on lines 240-241, were only major tryptic glycopeptides considered? This should be commented on.

Lines 273-274: What levels of prolactin were found in macroprolactin group? It would be useful to have these data for comparison, this information could be included in the Table 1.

Figures 2-3: Capital letters were used to mark panels on the figures (A-D), however, in the text with figure legends, small letters are used for these panels (a-d). Please, make it consistent in text and figures.

Line 120: The term of ‘immunobased’ is not correct please, use ’immune based’ .

Line 223-224: Patient group has significantly increased IgA, it should be mentioned that prolactin stimulates IgA production (1) and commented on.

Lines 294-295: Why the patients with macroprolactin (did you mean macroprolactinaemia patients?) were not included in correlation between prolactin and IgG2/3 galactosylation?

Line 363: Macroprolactin is a complex of immunoglobulin and prolactin. Was this macroprolactin captured together with other IgGs on the Protein G Sepharose column during immunoglobulin enrichment? If so, some of the analysed IgGs might be from this complex, this should be discussed. Macrolactin could be also an interesting object of follow up further investigation of glycomics changes and/or possible underlying glycan motif behind this complex formation associated with hyper/macroprolactinaenia.

Lines 356-362: IgG can also be O-glycosylated, so some sialic acid could come from there, this could be added to the discussion about sialylation.

Typos to be corrected: Line 243- older, line 341-menopause

Only IgG glycosylation was investigated, but the glycosylation on the other immunoglobulins maybe also affected in hyperprolactinaemia, this could be also included in the discussion.

References are not uniformed. Please, remove/or add all the DOI numbers after a referred publication.

Conclusion. After addressing these minor comments this manuscript will be suitable for publication in the PLOS ONE.

References.

1. Hermann G, O’Dorision MS. Modulation of IgA synthesis by neuroendocrine peptides. Trends Endocrinol Metab, 1991, 2(2): 68-72.

Reviewer #2: The authors examine the immunoglobulin G glycosylation pattern in patients with hyperprolactinaemia compared to healthy controls. The topic is interesting, unexplored and relevant, as it might provide insight into the relationship of this sex hormone with IgG glycome, more precisely, the proposed regulation of IgG glycosylation by estrogen. They examine in total 47 patients, divided into age- and sex-matched patient and control groups of similar subject number. They analyze the total IgG sialic acid content and subclass specific IgG Fc galactose and bisecting GlcNAc content. The results are more or less well presented, but I have a major issue with how the data were analyzed. Firstly, the patient group (25 subjects) appears way to small to be further divided into 3 subgroups. We are given no info on whether the subgroups are age- and sex- matched. Even if this were the case, this subdivision significantly increases the number of statistical tests performed. Which brings me to my second objection: no correction for multiple testing is performed. From the methods and results description, close to 30 statistical tests were performed and I am afraid some (if not all) p values deemed statistically significant might get lost after the correction. Unless there is a strong reason (is only a mild increase in prolactin level biologically relevant compared to very high values?), I would suggest only performing statistical analysis on the two main groups (patient vs. control) and re-assessing the results. Additionally, looking into the correlation of IgG glycosylation traits with prolactin levels on such a small sample number also seems problematic if age- and sex- correction is not previously performed on IgG glycan data. In summary, although the low sample number might not be sufficient to capture the small effect size that might be in effect here, I think this is an interesting pilot study and I believe it merits publication once the data is re-analyzed.

Minor issues:

- Some typos and inconsistencies in style.

- The common Fc glycopeptide of IgG2 and IgG3 subclass should be denoted as IgG2/3 at all times, to avoid ambiguity.

- Original glycan data (individual structures) not provided.

- Since the number of samples is small, I think the graphs would profit from single dot=subject representation in addition to characteristic descriptive values.

- Line 167: should it be “200 ug” instead of “200 mg”?

- Line 222: should it be “distributed” instead of “disturbed”?

- Line 338: I don’t think we can infer any causality from this study.

- A comment/question for the Discussion section: could correlation between prolactin levels and IgG glycoslyation traits be additionally “blurred” by prolactio concentration variation during menstrual cycle?

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Reviewer #1: Yes: Dr Radka Fahey (Saldova) and Dr Zsuzsanna Kovacs

Reviewer #2: No

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Altered immunoglobulin G glycosylation in patients with isolated hyperprolactinaemia

PONE-D-20-32839R1

Dear Dr. Landberg,

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.

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Kind regards,

Frederique Lisacek

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

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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: The authors implemented all suggested modifications. The authors' work had improved the quality of the manuscript and we support the acceptance of the manuscript for publication in the journal of PLOS ONE.

We have only one minor suggestion- the numbering of equations on lines 211 and 218 in the revised manuscript with tracked changes are rather confusing as they do not seem to be referenced in the text, are these labels necessary?

Reviewer #2: The authors have addressed all of my comments and I believe the manuscript is now ready for publication.

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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.

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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