From the journal RSC Chemical Biology Peer review history
The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR
Round 1
Manuscript submitted on 11 Нау. 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/chevron-down.png)
30-Mar-2021
Dear Dr Jimenez-Barbero:
Manuscript ID: CB-ART-03-2021-000051
TITLE: The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR
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. In particular, one reviewer has concerns over the treatment of the ITC data, which should be carefully revised.
Please submit a revised manuscript which addresses all of the reviewers’ comments. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.
Please submit your revised manuscript as soon as possible using this link:
*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***
https://mc.manuscriptcentral.com/rsccb?link_removed
(This link goes straight to your account, without the need to log on to the system. For your account security you should not share this link with others.)
Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.
You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.
Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.
RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.
The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.
Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id
Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.
I look forward to receiving your revised manuscript.
Yours sincerely,
Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
************
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-comments.png)
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/chevron-down.png)
Reviewer 1
A well thought out and well documented study, this report is at the cutting edge of NMR studies of protein-carbohydrate interactions. It provides detailed and convincing analyses of ligand recognition by two different domains of the same human galectin-8 protein. These studies have implications for how this key protein interacts with partners, in connection with infection/immunity. As such, this study is likely to be of general interest to the readership pf RSC Chemical Biology.
Reviewer 2
The authors provide a thorough characterization of the interaction of full length galectin-8 with several oligosaccharides of biological interest, completed by dissection of the two domains. This is an important work that complete our partial knowledge of a human lection of biological interest. The different biophysical approaches are well performed and the results are solid. Some clarifications are suggested and it is requested to revise the treatment of ITC data.
1 The rational for the choice of oligosaccharide ligands of Figure 1 is not fully justified. Why testing sialylaled TF antigen, and not sialyllactose, of siallylLacNAc ?
2 The literature about structural studies on galectin 8 is abundant, with no than 30 crystal structures domains complexed with different oligosaccharides. The fact that the structural basis of interaction with glycans are known could have been indicated and some comparison between the predicted binding modes and the one previously characterized could have been introduced in the discussion (even if not with identical oligosaccharides)
3 Recent review from Cagnoni et al (doi 10.1042/BST20200311) brings clear view on specificity, structure and function of galectin-8 and its two domains and should be cited in the introduction.
4 The authors state that full length Gal8 cannot be crystallized. What about the structure in ref 9 (4HAN) Some comparison should be made
5 Caracterisation of full length Galectin-8 and the confirmation that the two binding sites are not dependent is of high interset. In this view, it is of upmost importance to display the ITC of titration of compound 6 after saturation by compound 3 and reverse. In the present version, the thermograms are not displayed (neither in supp info nor in the main text). Showing them in the main text is recommended. Also the “pre-loaded” part is not clearly explained in header of Table 1
6 The Molecular Dynamics approach should be introduced (at least partly) in the experimental section. Only details should go to supp info.
7 ITC experiments are of good quality but some details are missing in the experimental session, and the data treatment revised
a. Temperature of experiments is not indicated
b. Are experiments performed in duplicate or triplicate ? standard deviation should be given in Table 1 ( and the number of digit for each values could be reduced
c. The stochiometry (n) should be given when experimentally determined, with standard deviation
d. For FigS19, if Gal-8B/ 5 has not been measured, the figure should indicate “not determined” (instead of no-binding)
e. For low affinity ligand (LaNAc, TF, less extent Galili) experiments were run with excess of ligand, which allows to reach the plateau. Since sigmoid shape cannot be obtained, the n value should be fixed to 1 (not indicated). As described in work by Turnbull & Daranas (doi: 10.1021/ja036166s.), Kd can be safely estimated, but there is no confidence on the value of enthalpy and entropy which is reflected in data with TF antigen. The reviewer would recommend to avoid including entropy and enthalpy values for low affinity ligands.
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-response.png)
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/chevron-down.png)
This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.
Dear Gonçalo,
Many thanks for your letter. We are happy with the comments and would like to thank the precise details and discussions raised by reviewer 2.
We have now prepared a new version taking into consideration the comments of the reviewer. Changes are highlighted in yellow. The detailed point-by-point answers are given below.
Many thanks for your help
With my warmest regards
Jesús
REVIEWER REPORT(S):
Referee: 1
Comments to the Author:
A well thought out and well documented study, this report is at the cutting edge of NMR studies of protein-carbohydrate interactions. It provides detailed and convincing analyses of ligand recognition by two different domains of the same human galectin-8 protein. These studies have implications for how this key protein interacts with partners, in connection with infection/immunity. As such, this study is likely to be of general interest to the readership pf RSC Chemical Biology.
Referee: 2
Comments to the Author
The authors provide a thorough characterization of the interaction of full length galectin-8 with several oligosaccharides of biological interest, completed by dissection of the two domains. This is an important work that complete our partial knowledge of a human lection of biological interest. The different biophysical approaches are well performed and the results are solid. Some clarifications are suggested and it is requested to revise the treatment of ITC data.
1. The rational for the choice of oligosaccharide ligands of Figure 1 is not fully justified. Why testing sialylaled TF antigen, and not sialyllactose, of siallylLacNAc ?
Yes. Good point. The choice of the carbohydrate portion of the T antigen (Gal1-3GalNAc) and of the 2,3-sialyl T antigen (Neu5Ac2-3Gal1-3GalNAc) was made on the basis of previous data that described higher-affinity and selective ligands for Gal-8N. Indeed, glycan array studies indicated that Gal-8N prefers 3’-sialylated/sulfated Galβ1-4Glc, type I LacNAc or Galβ1-3GalNAc, whereas type II LacNAc was less preferred (Carlsson et al., Glycobiology 2007, 17, 663–676). Therefore, we selected 2,3-sialyl-Galβ1-3GalNAc as high-affinity and high-selective ligand. The binding to Galβ1-3GalNAc was analyzed for comparison purposes. In contrast, the blood group determinants A and B were selected as the preferred described ligands for Gal-8C and therefore, LacNAc was used in this case as the minimal epitope reference. An explanatory sentence about the selection of the ligands has now been included in the main text.
2 The literature about structural studies on galectin 8 is abundant, with no than 30 crystal structures domains complexed with different oligosaccharides. The fact that the structural basis of interaction with glycans are known could have been indicated and some comparison between the predicted binding modes and the one previously characterized could have been introduced in the discussion (even if not with identical oligosaccharides).
Yes, we thank the reviewer for the suggestion. Although we mentioned that the proposed binding modes agreed with the shared binding for all galectins, we did not emphasized the fact that X-ray structures are also available for the CRD domains of Gal-8C and Gal-8C as well as Gal-8N in complex with simple carbohydrates. We have now included this information.
3 Recent review from Cagnoni et al (doi 10.1042/BST20200311) brings clear view on specificity, structure and function of galectin-8 and its two domains and should be cited in the introduction.
Yes, we completely agree with the reviewer. The corresponding review has now been cited as reference 16.
4. The authors state that full length Gal8 cannot be crystallized. What about the structure in ref 9 (4HAN). Some comparison should be made.
Yes, indeed four pdb entries are reported for Gal-8FL: 4HAN, 3VKL, 3VKM and 4FQZ. However, all X-ray crystal structures correspond to a mutant Gal-8FL, where the peptide linker between both domains has been replaced by a shorter histidine-methionine spacer. The employment of this mutant, more stable against protease activity and displaying lower flexibility allowed the X-ray structural analysis of Gal-8FL. Although this mutant isoform resulted active in neutrophil adhesion as its homologous Gal-8FL (N. Nishi et al., FEBS Lett. 2005, 579, 2058), the interplay between both CRDs or between the CRDs and the linker is likely to be profoundly different. These aspects were not investigated for the described mutant forms. Remarkably, a special orientation of both CRDs is defined in the NDP52–galectin-8 complex; however, the effects, if any, on the binding of the individual CRDs were not discussed. Nevertheless, in order to highlight all these aspects, a new sentence has now been included in the discussion section.
5 Caracterisation of full length Galectin-8 and the confirmation that the two binding sites are not dependent is of high interset. In this view, it is of upmost importance to display the ITC of titration of compound 6 after saturation by compound 3 and reverse. In the present version, the thermograms are not displayed (neither in supp info nor in the main text). Showing them in the main text is recommended. Also the “pre-loaded” part is not clearly explained in header of Table 1.
Yes, we thank the reviewer for noticing this aspect. The thermograms have been now included as Figure 2. Similarly, a more detailed explanation of the meaning of “pre-loaded” species has been included in the main text (header table 1) as well as in the supporting information. Indeed, the amount of ligand 3 or 6 required to saturate either the N-term CRD or the C-term CRD was based on the 15N-HSQC titration experiments, with 10 and 8 equivalents, respectively. These species, in which one single CRDs is saturated with its corresponding high-affinity ligand, were referred to as “pre-loaded”.
6 The Molecular Dynamics approach should be introduced (at least partly) in the experimental section. Only details should go to supp info.
The experimental details related to Molecular Dynamics (MD) simulations have been included in the experimental section.
7 ITC experiments are of good quality but some details are missing in the experimental session, and the data treatment revised.
Yes, we thank the reviewer for all their suggestions. We have added a more detailed description of the data acquisition and treatment in the experimental section.
a. Temperature of experiments is not indicated
The experiments were performed at 25 C
b. Are experiments performed in duplicate or triplicate ? standard deviation should be given in Table 1 ( and the number of digit for each values could be reduced.
The thermograms for each protein:ligand complex were acquired at least in duplicate and the data included in Table 1 resulted from the average between both independent measurements. Now, the data are shown as average standard deviation employing significant figures. When the standard deviation calculated from two independent measurements was lower than the instrument precision, the latter value was used to determine the accuracy of the final displayed data.
c. The stochiometry (n) should be given when experimentally determined, with standard deviation
Yes, we thank the reviewer for noticing these missing data. The experimentally determined n values have been included in the table, also with the corresponding standard deviation. N values were only determined for high-affinity ligands, or more precisely when ITC experiments were acquired at high values of c (c = [protein]/KD), c>1. Both data treatments (n was fitted or used as a fixed value) were performed in the case of low-affinity ligands (LacNAc, Galb1-3GalNAc and Galili). Remarkably, data arising from allowing n fitting was only employed when the experimentally determined n values were close to the expected ones. We employed n =1 in the case of Gal-8N and Gal-8C binding isotherms, and n = 2 in the case of Gal-8FL-LacNAc interaction since 15N-HSQC titration experiments revealed the involvement of both CRDs from the first titration points and therefore, it suggested the formation of 1:2 complexes. Although a similar behavior would be expected for Gal1-3GalNAc (with KD(Gal-8C) = 1.8 KD(Gal-8N)) and the formation of 1:2 complexes is highly likely, this is not the case for Galili (with KD(Gal-8N) = 3.8 KD(Gal-8C)). Thus, for the complex between Galili and Gal-8FL both KD values, for n= 1 and n=2, respectively, have been included (only determined values for n=1 were initially indicated). All details regarding data treatment have now been included in the experimental section.
d. For FigS19, if Gal-8B/ 5 has not been measured, the figure should indicate “not determined” (instead of no-binding)
Yes, we thank the reviewer for noticing this misleading aspect. The figure has been modified.
e. For low affinity ligand (LaNAc, TF, less extent Galili) experiments were run with excess of ligand, which allows to reach the plateau. Since sigmoid shape cannot be obtained, the n value should be fixed to 1 (not indicated). As described in work by Turnbull & Daranas (doi: 10.1021/ja036166s.), Kd can be safely estimated, but there is no confidence on the value of enthalpy and entropy which is reflected in data with TF antigen. The reviewer would recommend to avoid including entropy and enthalpy values for low affinity ligands.
We really thank the reviewer for the suggestion. Indeed, only in the case of the high-affinity ligands (when c >> 1), n values were experimentally determined. For low-affinity systems and when the “n” fitted values were far from the expected ones (n =1 or n=2), the n values were fixed. Now, only data arising from fixed n values have been used for LacNAc, Gal1-4GalNAc and Galili as indicated in the table and in the experimental section.
Regarding the inaccuracy of entropy and enthalpy values experimentally determined for low-affinity ligands, we completely agree with the remarks made by the reviewer. Indeed, we tried to avoid any discussion related with those values along the main text and no conclusions were extracted from their analysis. Thus, the values by themselves and/or any reference to enthalpy and entropy contributions have been removed and the corresponding explanatory note in the table together with the appropriate reference have been also included.
Round 2
Revised manuscript submitted on 09 Сәу. 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/chevron-down.png)
10-Apr-2021
Dear Prof Jimenez-Barbero:
Manuscript ID: CB-ART-03-2021-000051.R1
TITLE: The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR
Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.
You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with publication of your manuscript.
You can highlight your article and the work of your group on the back cover of RSC Chemical Biology, if you are interested in this opportunity please contact me for more information.
Discover more Royal Society of Chemistry author services and benefits here:
https://www.rsc.org/journals-books-databases/about-journals/benefits-of-publishing-with-us/
Thank you for publishing with RSC Chemical Biology, a journal published by the Royal Society of Chemistry – connecting the world of science to advance chemical knowledge for a better future.
With best wishes,
Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-comments.png)
![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/chevron-down.png)
Reviewer 2
All questions and suggestions have been answered. No further requests
Transparent peer review
To support increased transparency, we offer authors the option to publish the peer review history alongside their article. Reviewers are anonymous unless they choose to sign their report.
We are currently unable to show comments or responses that were provided as attachments. If the peer review history indicates that attachments are available, or if you find there is review content missing, you can request the full review record from our Publishing customer services team at RSC1@rsc.org.