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Dear Dr.  Van de Wetering

Thank you very much for submitting your Research Article entitled 'The membrane protein ANKH is critical for normal development and plays a key role in the cellular export of citrate and ATP from mammalian cells' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. We cannot, of course, promise publication at that time.

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Gregory Barsh

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: please see reviewer comments as attachment

Reviewer #2: This manuscript reports on some novel functions of the transmembrane protein ANKH/Ank. The most important findings include the transport of nucleoside triphosphate and the TCA cycle intermediates by ANKH/Ank.

Critique

1. A major concern is the authors' conclusion that ANKH/Ank does not transport pyrophosphate. The authors quote the original published report in the journal Science as the prior evidence for pyrophosphate transport via ANKH by other investigators. But subsequent studies seem to have established quite convincingly that ANKH does transport pyrophosphate using heterologous expression in canopus leaves oocytes. As this is one of the major issues focused in this manuscript, it would be nice of the authors could use their HEK293 cells over expressing wild type ANKH to monitor pyrophosphate uptake. Previous studies from other laboratories have shown that frog oocytes expressing ANKH mediate pyrophosphate uptake; the data from these studies seem convincing. It is possible that the conclusions reported in the current manuscript are correct, but this needs further verification.

2. With reference to the concern raised above, the discordance between PPi levels and AMP levels in the medium of HEK293 cells expressing ANKH is of signficance. PPi levels are twice that of AMP levels. Even when the levels of other NMPs are taken into account, still PPi levels are significantly higher than the cumulative levels of all NMPs combined. This discrepancy needs explanation. What if ANKH transports not only NTPs but also PPi? Would this explain the discrepancy?

3. Fig. 5. Urine from patients with ANKH mutation has no citrate whereas urine from and/ank mutant mice show significant levels of citrate. The authors attempt to explain this difference with potential differences in dietary citrate. This conclusion seems arbitrary. This would mean that the plasma of this particular patient had no citrate at all, thus explaining the absence of citrate in urine. is this true? Of course, the ankh/ank mice do show citrate in plasma and therefore, the authors could argue that urine shows citrate. But this will depend upon whether or not the same explanation is true for plasma from the human patient. For the authors' argument to be correct, the patient plasma should not have citrate. This seems very unlikely.

4. Other studies have shown that ANKH is expressed in kidney and in kidney epithelial cells. In fact, the protein is expressed in the plasma membrane of these cells. What could be the role of ANKH in renal handling of citrate and hence in the presence or absence of citrate in urine?

Minor comments

1. Page 4: ENPP1 is written as "ecto-nucleotidase pyrophosphatase/phosphodiesterase 1" It is "ecto-nucleotide pyrophosphatase/phosphodiesterase 1. This term has been used correctly in Authors Summary.

2. Page 4, second para, line 3: change "which" to "with".

3. Page 11: citrate is not found at 180 mM in sperm. This concentration should refer to seminal plasma, not sperm.

Reviewer #3: Szeri et al present some very interesting findings on the release of inorganic phosphate, citrate and to a lesser extent malate and succinate via membrane transporter protein ANKH. This reviewer is not an expert on bone function but the findings will be of great interest to investigators in many fields as plasma citrate has been implicated in ageing, type 2 diabetes, neuronal function and cancer but knowledge of its efflux from cells is incomplete. Similarly, as far as I am are the investigators are the first to provide evidence for a transporter of malate efflux in mammalian cells and plasma malate has been implicated in ageing and cardiovascular disease.

In general, the studies have been well conducted, the data look straightforward and convincing.

Results

Figure 1 Done in 293 cells but well controlled. The data look convincing but could the authors include an indication of the P values in the Figure or Figure legend at least for the final time point?

Figure 2 AMP highly significant. Is ENPP1 activity high though in relevant tissues such as bone and muscle for example, if so, this would add weight to the authors’ hypothesis that PPi comes from the conversion of NTPs to NMPs.

Figure 5 Line 207 the reference to succinate should I think read Figure 5C and what about malate?

Methodology

Could the authors give more details of the culture methods used and in particular whether the DMEM High clone medium and the antibiotics contained citrate? This is important because some suppliers provide antibiotics in citrate buffer.

LC/MS and NMR Can the authors distinguish between citrate and isocitrate with the methods they have used? If not this should be made clear in the methods section and in the discussion.

Discussion

This could be improved as it largely understates the importance of citrate in disease.

The citrate importer SLC13A5 knockout mouse is resistant to type 2 diabetes (Birkenfeld et al Cell Metabolism 2011) and essential for normal neuronal functioning in humans (Mycielska and Geissler Current Molecular Medicine 2015). Furthermore, citrate via pmCiC has been implicated in cancer growth (Mycielska et al 2018) and so these papers also add weight to the authors suggestion that extracellular citrate and ANKH could have a more widespread role in human pathologies and could be cited.

ANKH appears to mediate the secretion of malate and succinate as well as citrate. Although the levels of malate and succinate secretion are less then citrate, as far as I am aware there is no previous report of a mammalian malate transporter and this should be mentioned as it could explain the widespread reports of malate in human plasma.

Can the authors distinguish between citrate and isocitrate? These issues are important because citrate/isocitrate and phosphate have been reported to be two of the 22 metabolites linked to chronological age independently of BMI, blood pressure, fev, liver function and telomere length (Menni et al Int. J. Epidemiol. 2013) and isocitrate and malate are inversely associated with longevity and are associated with increased cardiovascular disease risk (Cheng et al Nature Communications 2015). Isocitrate is more significant than malate. These studies also support the authors speculation that ANKH might have widespread biological effects beyond bone.

Line 249 and reference 19 I think citrate in the plasma is normally much lower than 300µM in healthy people the range quoted in another review was 52-106 µM but it doubles in diseases like non-alcoholic fatty acid disease (Iacobazzi and Infantino Biol. Chem. 2014).

Line 269 The mechanism of citrate release by astrocytes is not known (Mycielska and Geissler Current Molecular Medicine 2015) and so do astrocytes express ANKH?

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes: Jirko Kühnisch

Reviewer #2: No

Reviewer #3: No

Attachments

Attachment

Submitted filename: 200319_Review_Plos_Genetics_ANKH_citrate.docx

Dear Dr. Van de Wetering,

We are pleased to inform you that your manuscript entitled "The membrane protein ANKH is crucial for bone mechanical performance by mediating  cellular export of citrate and ATP" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Summary

The presented revised manuscript describes metabolic abnormalities of malate/citrate and nucleotide metabolism. These findings are set into the context of Ankank/ank mice bone biology.

The reviewer is still highly positive for the novel finding of altered citrate levels after deactivation of ANKH in vitro and in vivo. This study could potentially provide first evidence linking general metabolism with ANKH associated phenotypes

The criticism of the reviewer was addressed by the authors. The authors add 3 supplementary figures. However, none of the presented data (figures) were significantly adapted or expanded to improve presentation, information and scientific basis of conclusion.

Major Criticism

The authors adapted the title of the manuscript. Now, the authors link altered citrate and ATP export to altered bone mechanical performance. This title is not justified by the presented mechanical and structural bone data. The structural bone data focus on cortical bone and are incomplete. The mechanical bone data are insufficient to identify differences between the collagenous, ECM and mineral phase. The accumulated bone and mechanical bone data cannot be linked to altered citrate levels. Moreover, the title suggests the cellular export of citrate and ATP directly impacts bone mechanics. For such a statement bone cells e.g. osteoblasts need to be analyzed.

The main source of systemic citrate are metabolic highly active tissues such as liver or skeletal muscle. Thus, the cause of altered systemic citrate levels was not clarified by the study. Mixing up Hek293 in vitro and incomplete bone data is not sufficiently answering the topic of the study. The study is not able to differentiate between local and systemic effects of altered metabolism

The authors statement “Our data indicate reduced levels of extracellular citrate at least partly underly the observed delay in osteogenic differentiation of Ankank/ank osteoblast.” is wrong. They did not analyze osteoblasts.

The authors statement “Possibly, also part of the ankylosis inhibitory effect of ANKH might come from citrate released into the joint space.” is highly speculative.

The discussion of the bone findings is superficial and not adequate to the manuscript title. The discussion of citrate in bone is not adequate to the literature. Role of citrate and bone (PMID 31731473).

Technical Criticism

Fig1A. Loading control of reference proteins were added. The authors did not improve quality of ANKH detection. Quantitative PCR to verify ANKH transcript levels in the generated cell lines was not added.

The authors comment on the cell approach e.g. by seeding 500 K cells/well. The authors did not normalize their generated data to the final cell number/protein content/ DNA amount/ ATP content. Presenting cell experimental without normalization is not state of the art.

The authors provide a statement where the measurement has been done. The authors did not improve the figure by adding images of the measured bone site and the observed phenotype. All these data are easily available and can be published.

The authors did not show the complete list of bone measurement parameters. This is not state of the art and makes the data not fully interpretable. The graphs do not use the standard abbreviation for microCT bone parameter. The material and methods do not provide the calibration approach for bone density measurement. What means the parameter eccentricity? Which bone perimeter value is used Ps.Pm or Ec.Pm?

The authors did not expand the data for bone mechanical analysis, which may help to differentiate effects of the ECM, collagenous matrix or mineral phase. These data are essential to identify the impact of altered PPi and citrate levels.

Reviewer #2: The authors have addressed all of the comments raised during the initial review.

Reviewer #3: The authors have addressed all of my questions satisfactorily.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes: Jirko Kühnisch

Reviewer #2: Yes: Vadivel Ganapathy

Reviewer #3: No

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