From the journal RSC Chemical Biology Peer review history

Berlin 14 December 2022

Dear Dr Jessen:

Manuscript ID: CB-ART-11-2022-000235
TITLE: The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. I received additional comments which I added below, which I found important to be addressed:

1) The authors have been very economical in their citation of the literature: 1) the citation of Sprigg et al 2022 as an example of anion exchange chromatography to separate inositol phosphates fails to mention that a spectrum of inositol phosphate species in kidney were shown to be responsive to dietary intervention in that study. Surely, those findings are more relevant to the discussion than the observation that anion exchange hplc exists.

2) Given the weight given to CE-MS as a method of separating inositol trisphosphates, here the quantification is exemplary, a discussion of the resolving power of other chromatographic approaches for inositol trisphosphates might be relevant. The authors have not been wholly candid about what has been achieved with other methods. Here the study of Freund et al. Eur. J. Biochem. 207, 359-367 (1992) is exemplary in its description of a protozoal phytase that produces Ins(1,2,3)P3. Mind you, so is the work of Murthy's group, 1996, https://doi.org/10.1104/pp.106.4.1489; Greiner et al. 2001, doi: 10.1016/s0168-1656(00)00331-x; 2006, doi: 10.1021/jf0100090; Lim and Tate, 1972, doi.org/10.1016/0005-2744(73)90160-5; and others, in identifying Ins(1,2,3)P3 and Ins(1,2,4)P3/Ins(1,2,6)P3. Similarly, Ins(1,2,3)P3 as an intermediate of phytase action in plants was reported Brearley and Hanke, 1996; 10.1042/bj3180279.
As we can see these are historic studies. That Ins(1,2,3)P3 does not figure in the modern reviews of the topic cited by the authors [refs 2,19-22] - reflects, perhaps, a lack of diligence on the part of the reviewers and can, hardly, be used to justify the novelty of the submitted work.

3) The characterization of the phytase employed here is rudimentary by the standards of phytase characterisation - Other much-better characterized phytases could, equally, have been used.



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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

************

Reviewer 1

In essence, this manuscript employs CE-MS and a recombinant protein to identify an inositol trisphosphate isomer in kidney stones. It does so effectively with all appropriate controls. This finding is novel. The enzyme employed is not well-characterized in this manuscript. The use of CE-MS for focused separation of inositol trisphosphates has not been described before.

What the manuscript fails to do, is put the biological relevance of this observation in context of kidney function or in context of the inositol phosphate speciation / metabolism of kidney (for which there is an extant literature). This is an omission.

The use of an enzyme, here a poorly-described phytase, to assist in stereoisomeric assignment is incisive - following a long-tradition of employ of (semi)-purified proteins/recombinant enzymes to characterize inositol phosphates extracted from tissues. The authors might consider citing other examples of use of enzymes or other procedures to assign identity to inositol (tris)phosphates, the literature is littered with examples and the approach is a critical facet of much of what we know about the inositol trisphosphate complement/biology of plants, fungi and animals.

The authors could choose to comment that of the twelve theoretical peaks of InsP3 (in a minor oversight, the covering letter incorrectly claims 41 possible isomers of InsP2-InsP3) resolvable on non-chiral chromatography, this study separates six peaks (Fig. 1C). How does this compare with other studies? The work of Mayr is relevant for non-radioactive methods and there are many studies that have used radiolabelling and adjunct enzymes, much like the phytase of the title, to achieve the same kind of outcomes.

If it is of merit to have identified Ins(1,2,3)P3, then a wider discussion of Ins(1,2,3)P3 and its occurrence in biology, of plants, animals and fungi is relevant. This inositol phosphate has been described with rigour as the product of plant alkaline phytase (Barrientos et al. 1994, https://doi.org/10.1104/pp.106.4.1489) with subsequent identification of the enzyme as a MINPP (Mehta et al. 2006). More generally, Ins(1,2,3)P3 and Ins(1,2,4)P3/Ins(1,2,6)P3 have been described as product(s) of phytases of plant, fungal, bacterial and protozoal origin. The studies of Lim and Tate, Cosgrove, Greiner, and Freund eg. (Eur. J. Biochem. 207, 359-367 (1992)) and others is relevant to the narrative.

Reviewer 2

CB-ART-11-2022-000235

This is an exciting manuscript looking at overlooked lower inositol phosphates. In general, the analytic chemistry of InsPs has been challenging and has hampered the understanding of their importance in biology, so methods to improve on this in relevant biological samples is important. Here, the authors used capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) to analyze various InsP2 and InsP3 isomers. They then convincingly used this method combined with clever hydrolysis products to identified the main InsP3 produced by the bacterial effector RipBL1. Interestingly, it turned out to be the less studied Ins(1,2,3)P3. Even more exciting is that Ins(1,2,3)P3 turns out to be an important constituant of kidneys stones and present in human urine samples.

I believe that this is a timely and important manuscript that should be accepted after minor revisions detailed below:

In fig. 1D, is Ins(1,3)P2 still co-eluting wih Ins(2,4)P2? Similarly, is Ins(1,4,6)P3 still under another peak in Fig 1D? If yes, they should be labeled as well.

Figure S2, please add the calculated m/z so that reader can compare with the observed m/z.

The authors may want to add references about inositol phosphates analogs as therapeutics (PMID: 32328427) when discussing the link between InsPs and kidney stones. Similarly, clinical trial against pathological calcification involving IP6 and analogs could be mentioned (PMID: 32024848, PMID: 35035944). These references would emphasize that the link between InsP and calcification is relevant.

I think that the authors mean Fig S7 in this sentence on p.9: “The most intense peak has an identical migration time with [13C6] Ins(1/3,2)P2 generated by pyrohydrolysis from [13C6] Ins(1,2,3)P3 (Figure S6), indicating Ins(1,2)P2 and/or Ins(2,3)P2 are present in kidney stones.”

I wonder if the InsP3 seen in kidney stone comes from a possible intracellular reservoir or if it is a product of hydrolysis of higher InsPs that may or may not be selected by chelation to the inorganic surface or serum calcium. The 2 position here might not be innocuous since it allows chelation because of cis phosphates esters in position 1/3-2. This could be discussed further.

Dear editor,

thank you for the input. I have uploaded a rebuttal letter.

with kind regards,

Henning Jessen

This text has been copied from the Microsoft Word response to reviewers and does not include any figures, images or special characters:

Dear Prof. Süssmuth,

We have resubmitted our corrected version of “The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones”. We thank the reviewers for their time and very valuable input and have made changes to the manuscript. These are highlighted in yellow in the main manuscript. I hope that our changes are addressing the reviewers concerns appropriately and we look forward to your decision.
Below, you will find a detailed reply to the reviewer´s comments.

Reviewer/Editor #1
1) The authors have been very economical in their citation of the literature: 1) the citation of Sprigg et al 2022 as an example of anion exchange chromatography to separate inositol phosphates fails to mention that a spectrum of inositol phosphate species in kidney were shown to be responsive to dietary intervention in that study. Surely, those findings are more relevant to the discussion than the observation that anion exchange hplc exists.

R: In this paper, we focus on the separation method and assignment of isomers of InsP2-3 in human kidney stone and urine samples. In our work, we did not characterize inositol phosphate profiles from human tissues or organs. This is the reason, we did not comment on the interesting inositol phosphate changes observed in the kidney of chicken subjected to different diets, with food untreated or treated with phytase. Sprigg et al. 2022 analyse inositol phosphate using an original HPLC-MS approach. We had included this citation for the discussion of the various methods to study inositol phosphates.

2) Given the weight given to CE-MS as a method of separating inositol trisphosphates, here the quantification is exemplary, a discussion of the resolving power of other chromatographic approaches for inositol trisphosphates might be relevant. The authors have not been wholly candid about what has been achieved with other methods. Here the study of Freund et al. Eur. J. Biochem. 207, 359-367 (1992) is exemplary in its description of a protozoal phytase that produces Ins(1,2,3)P3. Mind you, so is the work of Murthy's group, 1996, https://doi.org/10.1104/pp.106.4.1489; Greiner et al. 2001, doi: 10.1016/s0168-1656(00)00331-x; 2006, doi: 10.1021/jf0100090; Lim and Tate, 1972, doi.org/10.1016/0005-2744(73)90160-5; and others, in identifying Ins(1,2,3)P3 and
Ins(1,2,4)P3/Ins(1,2,6)P3. Similarly, Ins(1,2,3)P3 as an intermediate of phytase action in plants was reported Brearley and Hanke, 1996; 10.1042/bj3180279.

R: We thank the reviewer for this comment, the references (37-42) have been inserted in revised manuscript. We acknowledge that other phytases partially produce Ins(1,2,3)P3. However, we want to respectfully point out that there is a difference in analytically monitoring in vitro reactions as opposed to patient samples in a complex matrix (we have now added “in biological samples” to point out the context). Also, our main point is that we used the phytase to selectively produce Ins(1,2,3)P3, not a mixture of isomers. In the references pointed out by the reviewer, Ins(1,2,3)P3 was not the only isomer of InsP3 produced. Only some specificity of hydrolysis of InsP6 by alkaline phytase from Lily Pollen can be assumed but the purity of Ins(1,2,3)P3 (reference 38) is not described. To obtain useful internal references, high purity is important. RipBL1 described in this paper as a new bacterial effector phytase can produce Ins(1,2,3)P3 as the only isomer of InsP3 with high purity and selectivity (> 95%).

As we can see these are historic studies. That Ins(1,2,3)P3 does not figure in the modern reviews of the topic cited by the authors [refs 2,19-22] - reflects, perhaps, a lack of diligence on the part of the reviewers and can, hardly, be used to justify the novelty of the submitted work.

R: We respectfully disagree; we had no intention to justify novelty of our work by mentioning these reviews. In fact, we do acknowledge several times that Ins(1,2,3)P3 has been described in mammalian tissues (“Ins(1,2,3)P3 was described in mammalian cells more than 25 years ago [16, 24] and other inositol phosphates with a phosphate ester in the 2-position clearly exist. However, the interest in these isomers has faded over time, and recent literature is not reporting on them anymore so their roles remain unresolved”. Recent authoritative reviews/overviews do not mention ‘historic’ Ins(1,2,3)P3 papers. We would like to point out that there are several of these reviews and leading players in the field (e.g. Shears, Irvine, Kim, and York) wrote them. Such papers are usually starting points for many young researchers entering the field. Pointing out that the Ins(1,2,3)P3 isomer was not mentioned in any of these reviews is therefore important. The citation of such reviews aimed to highlight the importance to study inositol phosphate signaling in its full complexity and not just focusing on the usual suspects (Ins(1,4,5)P3, Ins(1,3,4,5,6)P5, or InsP6) and pathways as reported in the cited reviews.

3) The characterization of the phytase employed here is rudimentary by the standards of phytase characterisation - Other much-better characterized phytases could, equally, have been used.

R: In our work, RipBL1 has been used to assign the isomeric identity of the kidney stone/urine component. Other phytases may have been used before but we decided not to use them, in large part, because RipBL1 selectively produces the molecule under investigation. Conversely, the previously published phytases generate a mixture of several compounds. Our objective was not the full characterization of RipBL1. A follow-up paper comparing RipBL1 to several other phytases will focus more on its systematic characterization.

Reviewer #2
Comments to the Author
In essence, this manuscript employs CE-MS and a recombinant protein to identify an inositol trisphosphate isomer in kidney stones. It does so effectively with all appropriate controls. This finding is novel. The enzyme employed is not well-characterized in this manuscript. The use of CE-MS for focused separation of inositol trisphosphates has not been described before.
What the manuscript fails to do, is put the biological relevance of this observation in context of kidney function or in context of the inositol phosphate speciation / metabolism of kidney (for which there is an extant literature). This is an omission.

R: We thank the reviewer for the positive comments. We would like to point out that we developed new protocols to detect lower phosphorylated InsP species with outstanding sensitivity to study kidney stones and urine from human patients. Our original discovery that noncanonical InsP3 isomers are present in kidney stones is opening new investigative possibilities. We agree with the reviewer that the biological relevance of our discovery has not been fully investigated. Kidneys are essential to regulate phosphate homeostasis, a process controlled by specific inositol phosphate in both yeast and plants. Additionally, the recent Sprigg et al. 2022 manuscript (see reviewer 1 point1) demonstrates that inositol phosphate metabolism in chicken kidneys are responsive to dietary intervention. Therefore, it is our uttermost intention to expand these observations in the future by dissecting mammalian kidney inositol phosphate metabolism and signaling. With the current knowledge, it will be very speculative to draw any biological conclusions about specific InsPs isomer's function in the kidney as we studied stones and urine.

The use of an enzyme, here a poorly-described phytase, to assist in stereoisomeric assignment is incisive - following a long-tradition of employ of (semi)-purified proteins/recombinant enzymes to characterize inositol phosphates extracted from tissues. The authors might consider citing other examples of use of enzymes or other procedures to assign identity to inositol (tris)phosphates, the literature is littered with examples and the approach is a critical facet of much of what we know about the inositol trisphosphate complement/biology of plants, fungi and animals.
R: We thank the reviewer for this comment, references (37-42) have been inserted in revised manuscript.

The authors could choose to comment that of the twelve theoretical peaks of InsP3 (in a minor oversight, the covering letter incorrectly claims 41 possible isomers of InsP2-InsP3)
R: We thank the reviewer for the comment on the cover letter. Indeed, it should have read 41 possible isomers for InsP1-InsP3).

resolvable on non-chiral chromatography, this study separates six peaks (Fig. 1C). How does this compare with other studies? The work of Mayr is relevant for non-radioactive methods and there are many studies that have used radiolabelling and adjunct enzymes, much like the phytase of the title, to achieve the same kind of outcomes.

R: In this paper, six InsP2 were separated and seven InsP3 (Figure 1B, 1C and 1D) could be separated by using two BGE buffers, respectively. Excluding all enantiomers, there are theoretically 9 InsP2 and 12 InsP3. Not all of them are commercially available and we have therefore not analysed all possible isomers. To the best of our knowledge, the separation of 6 InsP2 and 9 InsP3 is achievable by ion chromatography protocols (reference 19). The separation ability of our developed method is comparable to that. However, the quality of analytical methods is not only judged by the resolving power; an additional uttermost important parameter is also the sensitivity. Ion chromatography coupled with post-column iron staining results in insensitive detection and is, therefore, employed only for in vitro studies (reference 19). The limit of detection of our method, 1.3 femtomol for InsP2 and 800 attomol for InsP3, is by far the lowest compared to other chromatography-related protocols summarized in reference 35. A statement has been added in the text clarifying this point.

If it is of merit to have identified Ins(1,2,3)P3, then a wider discussion of Ins(1,2,3)P3 and its occurrence in biology, of plants, animals and fungi is relevant. This inositol phosphate has been described with rigour as the product of plant alkaline phytase (Barrientos et al. 1994, https://doi.org/10.1104/pp.106.4.1489) with subsequent identification of the enzyme as a MINPP (Mehta et al. 2006). More generally, Ins(1,2,3)P3 and Ins(1,2,4)P3/Ins(1,2,6)P3 have been described as product(s) of phytases of plant, fungal, bacterial and protozoal origin. The studies of Lim and Tate, Cosgrove, Greiner, and Freund eg. (Eur. J. Biochem. 207, 359-367 (1992)) and others is relevant to the narrative.

R: References (37-42) were inserted in revised manuscript (“Dephosphorylation of InsP6 by different types of phytases to produce Ins(1,2,3)P3, either as the final product or as intermediates, has been investigated.[37-41] Ins(1,2,3)P3 as an intermediate in barley aleurone tissue was described as well(42)”). We want to respectfully point out that we have discussed previous identification in mammalian cell lines: “Ins(1,2,3)P3 was described in mammalian cells more than 25 years ago.” This paper, as the reviewer has noticed, focuses on the CE-MS method development for InsP2-3 and assignment of isomers of InsP3 in patient samples (kidney stone and urine). We demonstrate for the first time that inositol phosphates are present in kidney stones and specific isomers are found in urine. Our objective was not the full dissection of RipBL1 activity. We use RipBL1 just as a tool to generate Ins(1,2,3)P3 (see answer to reviewer 1 point 3). It is too speculative to comment on phytase diversity without experimentally comparing RipBL1 to other phytases. We hope that the reviewer will understand our point.

Reviewer #3
Comments to the Author CB-ART-11-2022-000235

This is an exciting manuscript looking at overlooked lower inositol phosphates. In general, the analytic chemistry of InsPs has been challenging and has hampered the understanding of their importance in biology, so methods to improve on this in relevant biological samples is important. Here, the authors used capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) to analyze various InsP2 and InsP3 isomers. They then convincingly used this method combined with clever hydrolysis products to identified the main InsP3 produced by the bacterial effector RipBL1. Interestingly, it turned out to be the less studied Ins(1,2,3)P3. Even more exciting is that Ins(1,2,3)P3 turns out to be an important constituent of kidneys stones and present in human urine samples.

R: We thank the reviewer for this very positive evaluation of our work.

I believe that this is a timely and important manuscript that should be accepted after minor revisions detailed below:

In fig. 1D, is Ins(1,3)P2 still co-eluting wih Ins(2,4)P2? Similarly, is Ins(1,4,6)P3 still under another peak in Fig 1D? If yes, they should be labeled as well.

R: In Figure 1D, there was no Ins(1,3)P2. The separation of Ins(1,5)P2 and Ins(2,4)P2 was resolved using BGE of 50 mM ethylamine titrated with formic acid to pH 10.0, and the separation of Ins(1,3)P2 and Ins(2,4)P2 did not change too much as compared to using BGE of 35 mM ammonium acetate titrated with ammonium hydroxide to pH 9.75. For this, we do not show the data.
There was also no Ins(1,4,6)P3 in Figure 1D. Ins(1,4,6)P3 still coeluted with Ins(1,4,5)P3 (data not shown in the manuscript). A statement has been added.

Figure S2, please add the calculated m/z so that reader can compare with the observed m/z.
R: We have added the calculated m/z in the revised manuscript.

The authors may want to add references about inositol phosphates analogs as therapeutics (PMID: 32328427) when discussing the link between InsPs and kidney stones. Similarly, clinical trial against pathological calcification involving IP6 and analogs could be mentioned (PMID: 32024848, PMID: 35035944). These references would emphasize that the link between InsP and calcification is relevant.

R: This is indeed highly relevant! The references (48-50) have been inserted. A statement has been added: (“Recently, InsP6 analogues with PEG modifications were reported to completely inhibit such crystallization processes in the nanomolar range.[49] Additionally, studies with SNF472 (a hexasodium salt of InsP6) as an inhibitor of vascular calcification in a phase 2 clinical trial are ongoing,[50] highlighting a strong relationship between InsP6 and calcification.”)

I think that the authors mean Fig S7 in this sentence on p.9: “The most intense peak has an identical migration time with [13C6] Ins(1/3,2)P2 generated by pyrohydrolysis from [13C6] Ins(1,2,3)P3
(Figure S6), indicating Ins(1,2)P2 and/or Ins(2,3)P2 are present in kidney stones.”

R: We thank the reviewer for catching this! We have corrected it.

I wonder if the InsP3 seen in kidney stone comes from a possible intracellular reservoir or if it is a product of hydrolysis of higher InsPs that may or may not be selected by chelation to the inorganic surface or serum calcium. The 2 position here might not be innocuous since it allows chelation because of cis phosphates esters in position 1/3-2. This could be discussed further.

R: This is an excellent point. Kidney stone formation is not fully understood but one key mechanism is crystallization from urine as a result of supersaturation. As shown in the paper, the InsP3s observed in urine samples pretty much mirror the InsP3s observed in the stone (figures 3B and 4A). From the urine samples, we already removed any epithelial cells by centrifugation, indicating that the InsP3s might not come from a potential intracellular reservoir. According to the data we have so far, the general InsPs distribution in kidney stone (Figure 3B) show a similar trend with InsPs distribution in urine samples (Figure 4A). Thus, it seems that the InsPs are not preferentially selected and accumulated to a large extent by chelation to the surface. We have added a statement in the text.

On behalf of all authors, I would like to thank you for your helpful suggestions to improve the paper and for reconsidering our submission.

With kind regards,

Henning Jessen

Berlin, 26 January 2023

Dear Dr Jessen:

Manuscript ID: CB-ART-11-2022-000235.R1
TITLE: The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones

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Reviewer 1

This reviewer thanks the authors for prompt revision of the manuscript. The manuscript remains a statement of the art of the most sensitive detection of inositol phosphates. The assignment of identity to the species detected has involved careful employ of chemo/enzymatic synthesis of standards performed to an exemplary standard. The measurement of inositol phosphates without recourse to radiolabelling is undoubtedly very powerful and the arguments made carry weight.

The biological relevance of the findings is left to others to explain and explore further, but pointers to literature sources where the reader may find further insight are included. Here, the addition of extra references makes for a more balanced article.

This reviewer recommends publication.

Reviewer 2

I am satisfied with the revisions made to the manuscript. This work pushes the boundaries of what can be done in biological samples and reveals the presence of specific lower inositol phosphates in kidneys stones and human urine samples. I believe that the authors have also addressed the other reviewers comments by adding citations and clarifying the distinguishing features of this work compared to previous literature. I therefore recommend publication in RSC Chemical Biology.