From the journal RSC Chemical Biology Peer review history

25-Feb-2021

Dear Dr Aebi:

Manuscript ID: CB-ART-01-2021-000019
TITLE: Glycan-Protein Interactions Determine Kinetics of N-Glycan Remodeling

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.

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.

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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.

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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

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

Reviewer 1

Prof. Aebi and co-workers study how glycan-protein interactions determine the kinetics of N-glycan remodeling. For this purpose, they use the protein disulfide isomerase (PDI), which is a model protein with five potential glycosylation sites. They observed that glycan processing at the five PDI sites occurs with different kinetics for the four enzymes employed (ER Man I, GM I, GnT I, and GM II). Therefore, identical glycans at various glycosylation sites present different substrate properties to the processing enzymes. To understand these outcomes at the atomic level, they perform extensive MD simulations on glycosylated PDI and conclude that glycan conformation and accessibility on the protein surface can explain the kinetics data. According to the simulations, and in agreement with the experiments, site 4 of PDI was processed slowest by all studied enzymes.
The manuscript is clear, well-written and the results are relevant for the readership of the journal.
Major points:
1) According to the text, full-length PDI protein was used to model the protein with glycans at sites 1-4. However, when site 5 was analyzed, only the a'-domain was considered in the calculations. What is the reason for this?
2) The authors should also study by MD simulations the conformational changes prompted by reducing or alkylating the PDI protein. Why do these modifications improve the processing of the glycan at site 4?
3) Some MS spectra should be shown in the supplementary information.

Minor points:
1) In agreement with the PDB structure of PDI (2B5E), the glycan should be attached to Asn82 (site 1), Asn177 (site 2), Asn155 (site 3), site Asn174 (site 4), and Asn425 (site 5). However, the authors indicate Asn151 and Asn170 for sites 3 and 4, respectively. Is this correct?
2) The authors should explain how they obtain a total of 75 us for the full-length glycoprotein with glycosylated sites 1-4 and 110 us for the a'-domain with glycosylated site 5.
3) Figure S1: it would be interesting to show the structural ensembles instead of a frame obtained from the MD simulations. This should be done for all sites.

Reviewer 2

The manuscript by Mathew et al is an elegant biochemical study into the kinetics of glycan remodeling by different enzymes using one glycoprotein (PDI) with 5 different glycan sites. The biochemical evidence is strengthened by MD simulations of the accessibility of the different glycans, and aims to explain the glycan heterogeneity observed in glycan processing in the ER and Golgi. I believe this is an thorough study of interest to the readership of RSC Chem Biol, and would suggest accepting with major textual revisions and clarifications.

My main concern is about the connection between the MD simulations and the biochemical evidence. The MD simulations are presented in a lot of detail, focusing on 4 categories of accessibility, and the fraction of the different branches (A,B and C) of each glycan that fall in each category. Only very little attention is paid to the concave and convex surfaces. In contrast, the discussion section details mostly about accessibility based on the concave and convex surfaces, and only marginally links back to the 4 categories of accessibility. Moreover, the discussion is only about the difference between sites, and does not mention anything about the different branches. In my opinion, the manuscript would highly benefit from a more balanced discussion, where the MD results are better represented in the explanation of the biochemical results. By starting the results section with such a detailed description of branch modeling, the reader expects that this level of detail is also mirrored in the discussion. Is it possible to link specific enzyme activity (at a certain branch) to its accessibility as predicted by MD? Are the authors able to speculate on the impact of branch accessibility to enzyme activity?

Other textual edits:

Abstract: rephrase ‘differential, tertiary structure context dependent N-glycan’ for better readability

Page 3. ‘In the following’ sentence is missing some words

Page 4, section starts with ‘Understanding site-specific…’
- Refer to Figure 1 early on
- Start here with a better link to figure S2A, which actually has a lot of information that is very important to the manuscript! Explain the central role of the 4 enzymes, that are also under study here. Make explicit distinction between 1-step and 2-step enzymes.
- ‘same glycoside hydrolase family’, specify which family

Page 4, section start with ‘The resulting…’
- It is not clear how this ‘results’ from the previous section, so more clearly explain how Man5GlcNAc2 is produced, by which consecutive enzyme steps.
- Define ‘hybrid glycans’
- Define ‘complex glycan structures’
- End this section with a clear statement of the challenges/open questions in the field, to set the stage for the description of the manuscript (‘we used yeast protein…’)

Page 6, section start with ‘We simulated’
- Specify the inclusion of solvent here

Page 6, Conformations of PDI glycans
- The definition of the 4 categories is quite confusing, and would benefit from a more structured definition, maybe even with a schematic.
- Here a mention of solvent is made, but not specified (I expect water?)
- ‘GH47 a-mannosidases, such as ER Man 1,’

Page 7, section starts with ‘The accessibility’
- This section would benefit from more immediate references to the bars in Fig 1C. Now the reader really has to search for the right spot to see the data.
- ‘a considerable amount of exposed microstates’, please refer to specific data
- Also, clearly define what the bar ‘any’ means
- ‘A generally minimal exposure of the B branch IS related to its central location…’ I suggest to rephrase to ‘may be related…’
- ‘had a tendency to branch exposure but contacting conformations on sites 2 and 5’, rephrase for clarity of reading

Page 8: ‘surrounding protein surface topoly’ rephrase for clarity

Page 9, experimental set-up: briefly mention challenges associated with producing well-defined mammalian glycoproteins. Also describe the production of uniform glycoproteins, or if the authors consider this standard experiments, refer to prior publications describing the technique.

Page 9, ‘site-specific processing of N-glycans’
- Explain the choice for end-point kinetics
- Add info on time-frame for each enzyme
- For GnT1, one could argue that the conversion of site 4 may exceed the other if the reaction is left for longer time? Again, explain initial rate vs end-point

Page 11, section starts with ‘For PDI’
- Repeat the expected activity of GM1, and link to Fig S2A
- Are the authors able to define from which branch the first Man is removed, and then link this also to accessibility as modeled in MD?

Page 11, section start with ‘The global fits’
- why exclude the fit for site 4? In my opinion, the global fits for site 4 in Fig 4A also seem reasonable.
- Also the fit for Man6GlcNAc2 seems to have large deviations. The authors should better define what is meant with good or bad fit

Page 11, section starts ‘As shown above’
- more specifically introduce the issue with the ‘affected mannose trimming step’ by explaining that there are two steps
- I suggest to add at the end of this section ‘and virtually no final product GlcNAcMan3GlcNAc2 was produced.’

Page 13, ‘we demonstrated’
- ‘for a given enzyme’, rephrase to make this more explicit and explain again why the 4 enzymes under study are so interesting

Page 13, section starts with ‘We used MD simulations’
- sentence ‘ An effective enzyme accessibility…binding modes.’ is too long and unclear, please rephrase for readability
- ‘when binding to a-mannosidase’, can the authors specify which mannosidase?

Page 14, top section
- ‘structural differences of ER Man 1 and GnT 1’ define these differences, or link to a suitable reference

Page 14, section start with ‘ We perturbed’
- ‘We perturbed glycan-protein interactions…’ I don’t think the data convincingly shows that the interactions were indeed perturbed by reduction and alkylation, or at least the authors do not give a lot of proof here. I suggest rephrasing this to better reflect the small changes observed in kinetics of the alkylated proteins, as the authors also do a few sentences later.
- ‘Similarly’ is in my opinion the wrong connector, as denaturing is not similar to alkylation.

Figure 1C. y axis should be ratio. the bar called ‘any’ is confusing.

Legend Figure 2B: It is confusing what is plotted here, is it conversion/consumption of starting material Man9GlcNAc2, or production of product Man5GlcNAc2, as the y-axis suggests? Are there any intermediates observed, or is it a clean conversion from Man9 to Man5? Same question holds for Figure 2D.

Figure 5B. I suggest to use different tick symbols, as it is quite difficult to distinguish between native and red/alkyl. Or make clear which one is dashed and solid lines.

We thank the two reviewers for their very detailed and insightful review. With their input, we were able to improve our manuscript and we hope that our study can now be published in RSC Chemical Biology.

************
REVIEWER REPORT(S):
Referee: 1

Comments to the Author
Prof. Aebi and co-workers study how glycan-protein interactions determine the kinetics of N-glycan remodeling. For this purpose, they use the protein disulfide isomerase (PDI), which is a model protein with five potential glycosylation sites. They observed that glycan processing at the five PDI sites occurs with different kinetics for the four enzymes employed (ER Man I, GM I, GnT I, and GM II). Therefore, identical glycans at various glycosylation sites present different substrate properties to the processing enzymes. To understand these outcomes at the atomic level, they perform extensive MD simulations on glycosylated PDI and conclude that glycan conformation and accessibility on the protein surface can explain the kinetics data. According to the simulations, and in agreement with the experiments, site 4 of PDI was processed slowest by all studied enzymes.
The manuscript is clear, well-written and the results are relevant for the readership of the journal.

Major points:
1) According to the text, full-length PDI protein was used to model the protein with glycans at sites 1-4. However, when site 5 was analyzed, only the a'-domain was considered in the calculations. What is the reason for this?
The required computational resources for MD simulations typically grow with the total number of atoms N with a scaling of O(N log(N)) including the atoms of the protein and the explicit water molecules. Therefore, we minimized the number of water molecules in two ways. First, simulating the full-length PDI only with glycosylated sites 1-4 reduces the required box length towards the C-terminal domain which, in turn, reduced the number of total water molecules in the simulations. We checked that the glycans on site 1-4 are far enough from site 5 such that removal and separate simulation of site 5 on the a’-domain does not neglect mutual interactions between the two glycosylated surface regions. Second, we applied the NDLP box size optimization, which considerably reduced the number of required water molecules in the full-length PDI simulations as described in the SI.
2) The authors should also study by MD simulations the conformational changes prompted by reducing or alkylating the PDI protein. Why do these modifications improve the processing of the glycan at site 4?
We thank the reviewer for this interesting question. Unfortunately, we cannot answer it within the scope of this paper for two reasons. Most importantly, the structure and fold of the reduced and alkylated PDI are unknown such that we could not even speculate by conformational comparison. Further, the lack of such a structure in the PDB renders the proper modeling and simulations unfeasible as an experimentally determined starting point is crucial for protein MD simulations. Last but not least, the aggregate sampling in the manuscript required one year to generate on high-performance computing clusters. Hence, the additional simulations needed to answer the reviewer’s question go far beyond the scope of our presented work.
3) Some MS spectra should be shown in the supplementary information.
We added the MS/MS spectra of the five PDI glycopeptides to the Supplementary as well as the XIC of an exemplary in vitro assay which shows the decrease of one glycoform and the increase of another on site 4.
Minor points:
1) In agreement with the PDB structure of PDI (2B5E), the glycan should be attached to Asn82 (site 1), Asn177 (site 2), Asn155 (site 3), site Asn174 (site 4), and Asn425 (site 5). However, the authors indicate Asn151 and Asn170 for sites 3 and 4, respectively. Is this correct?
We thank the reviewer for spotting this important detail. This was only an error in the manuscript, which is corrected now.
2) The authors should explain how they obtain a total of 75 us for the full-length glycoprotein with glycosylated sites 1-4 and 110 us for the a'-domain with glycosylated site 5.
We thank the reviewer for this helpful suggestion. We added the detailed information in the Methods and the Results section. Individual simulations on the order of hundreds of nanoseconds were carried out in parallel, resulting in the total aggregate sampling of 75 us and 110 us, respectively.
3) Figure S1: it would be interesting to show the structural ensembles instead of a frame obtained from the MD simulations. This should be done for all sites.
We found this to be a valuable comment and thank the reviewer for it. We included an additional illustration of the conformational ensemble of the N-glycans on each site in the newly added figures S2 and S3.



Referee: 2

Comments to the Author
The manuscript by Mathew et al is an elegant biochemical study into the kinetics of glycan remodeling by different enzymes using one glycoprotein (PDI) with 5 different glycan sites. The biochemical evidence is strengthened by MD simulations of the accessibility of the different glycans, and aims to explain the glycan heterogeneity observed in glycan processing in the ER and Golgi. I believe this is an thorough study of interest to the readership of RSC Chem Biol, and would suggest accepting with major textual revisions and clarifications.

My main concern is about the connection between the MD simulations and the biochemical evidence. The MD simulations are presented in a lot of detail, focusing on 4 categories of accessibility, and the fraction of the different branches (A,B and C) of each glycan that fall in each category. Only very little attention is paid to the concave and convex surfaces. In contrast, the discussion section details mostly about accessibility based on the concave and convex surfaces, and only marginally links back to the 4 categories of accessibility. Moreover, the discussion is only about the difference between sites, and does not mention anything about the different branches. In my opinion, the manuscript would highly benefit from a more balanced discussion, where the MD results are better represented in the explanation of the biochemical results. By starting the results section with such a detailed description of branch modeling, the reader expects that this level of detail is also mirrored in the discussion. Is it possible to link specific enzyme activity (at a certain branch) to its accessibility as predicted by MD? Are the authors able to speculate on the impact of branch accessibility to enzyme activity?

We would like to thank the reviewer for this important comment and agree that the discussion is lacking a clear connection between the MD simulations of branch accessibility and the results obtained in the in vitro assays. Unfortunately, such a clear connection cannot be discussed, because the analytical method (MS) used for the in vitro assays does not provide any information about glycan isomers. Therefore, we can for example distinguish a Man7GlcNAc2 glycan from a Man6GlcNAc2 glycan (based on their different mass) but cannot determine from which branch the mannose was removed. It is thus not possible to directly compare information obtained by MD simulations about branch accessibility with the biochemical results obtained by enzyme in vitro assays at the same level of detail. However, we agree that this should have been more clearly explained and discussed in the manuscript. We therefore added a paragraph to the Discussion section describing these limitations in detail.


Other textual edits:

Abstract: rephrase ‘differential, tertiary structure context dependent N-glycan’ for better readability
We rephrased this complicated sentence for more clarity.

Page 3. ‘In the following’ sentence is missing some words
We rewrote the sentence.

Page 4, section starts with ‘Understanding site-specific…’
- Refer to Figure 1 early on
- Start here with a better link to figure S2A, which actually has a lot of information that is very important to the manuscript!
A sentence (“While being transported from the ER through the cis-, medial, and trans-Golgi, a glycoprotein encounters a different set of glycan processing enzymes in each compartment for a limited amount of time. Hereby, the product of an enzyme acting earlier in the pathway serves as a substrate for the enzyme acting afterwards” describing what can be seen in (now after revision) figure S4A was added here.
Explain the central role of the 4 enzymes, that are also under study here.
A sentence describing the importance and central role of the four enzymes used in this study was added.
Make explicit distinction between 1-step and 2-step enzymes.
We adapted the text to highlight the difference between enzymes performing a single trimming or transferase reaction (like ER Man I and GnT I respectively) and enzymes performing multiple mannose trimming steps (GM I and GM II).
- ‘same glycoside hydrolase family’, specify which family
We specified the glycoside hydrolase family in the text.

Page 4, section start with ‘The resulting…’
- It is not clear how this ‘results’ from the previous section, so more clearly explain how Man5GlcNAc2 is produced, by which consecutive enzyme steps.
The section was re-written for clarity.
- Define ‘hybrid glycans’
A definition for ‘hybrid glycans’ was included in the sentence.
- Define ‘complex glycan structures’
A definition for ‘complex glycan structures’ was included in the sentence.
- End this section with a clear statement of the challenges/open questions in the field, to set the stage for the description of the manuscript (‘we used yeast protein…’)
A paragraph describing the open questions in the field was added.

Page 6, section start with ‘We simulated’
- Specify the inclusion of solvent here
The use of explicit-water MD simulations is now mentioned on page 6.

Page 6, Conformations of PDI glycans
- The definition of the 4 categories is quite confusing, and would benefit from a more structured definition, maybe even with a schematic.
The description of the four categories now includes the corresponding references to the Fig. S1 and the Method section about the accessibility classifications.
- Here a mention of solvent is made, but not specified (I expect water?)
To reduce potential confusions we switched the wording ‘solvent’ to ‘water’.
- ‘GH47 a-mannosidases, such as ER Man 1,’
We included the side reference to ER Man I.

Page 7, section starts with ‘The accessibility’
- This section would benefit from more immediate references to the bars in Fig 1C. Now the reader really has to search for the right spot to see the data.
- ‘a considerable amount of exposed microstates’, please refer to specific data
- Also, clearly define what the bar ‘any’ means
Considering the three above mentioned comments, we introduce more carefully how the histograms are composed, including a description of the bar ‘any’. Notes on particular colors and features of the plots are placed in brackets in the text where it seems helpful to identify the specific data.
- ‘A generally minimal exposure of the B branch IS related to its central location…’ I suggest to rephrase to ‘may be related…’
We adopted the more careful formulation suggested by the reviewer.
- ‘had a tendency to branch exposure but contacting conformations on sites 2 and 5’, rephrase for clarity of reading
We split this sentence into three separate ones.

Page 8: ‘surrounding protein surface topoly’ rephrase for clarity
We rephrased and separated the sentence into two for clarity.

Page 9, experimental set-up: briefly mention challenges associated with producing well-defined mammalian glycoproteins. Also describe the production of uniform glycoproteins, or if the authors consider this standard experiments, refer to prior publications describing the technique.
A paragraph describing the challenges and the production procedures of well-defined glycosubstrates for the different enzyme in vitro assays was added to the main text.

Page 9, ‘site-specific processing of N-glycans’
- Explain the choice for end-point kinetics
- Add info on time-frame for each enzyme
A sentence addressing both points above was added.
- For GnT1, one could argue that the conversion of site 4 may exceed the other if the reaction is left for longer time? Again, explain initial rate vs end-point
To investigate this further we determined initial velocities for GnT I in the next section (Michaelis-Menten analysis of processing kinetics). A sentence that refers to this section was added.

Page 11, section starts with ‘For PDI’
- Repeat the expected activity of GM1, and link to Fig S2A
The expected activity of GM I was described again and readers are referred to figure S2A (now after revision figure S4A).
- Are the authors able to define from which branch the first Man is removed, and then link this also to accessibility as modeled in MD?
Because of the analytical method (MS) used in this study we are not able to identify from which branch the first mannose was removed. The limitations of this analytical method are briefly mentioned a bit later in the section “Rate constants describing appearance and disappearance of intermediate structures” on page 12 with the sentence “…
but we consider the results underdetermined, because we could not experimentally distinguish the different Man6GlcNAc2 isomers.”

Page 11, section start with ‘The global fits’
- why exclude the fit for site 4? In my opinion, the global fits for site 4 in Fig 4A also seem reasonable.
We thank the reviewer for this comment as the fit for site 4 should not have been excluded, we corrected the sentence.
- Also the fit for Man6GlcNAc2 seems to have large deviations. The authors should better define what is meant with good or bad fit
We agree that the fit for Man6GlcNAc2 has large deviations. We hypothesize that this is due to the presence of a second Man6GlcNAc2 isomer and discuss this issue in detail in the supplementary (see figure S9)

Page 11, section starts ‘As shown above’
- more specifically introduce the issue with the ‘affected mannose trimming step’ by explaining that there are two steps
We added a sentence describing the two different mannose trimming steps to this section.

- I suggest to add at the end of this section ‘and virtually no final product GlcNAcMan3GlcNAc2 was produced.’
The sentence was added to the end of the section.

Page 13, ‘we demonstrated’
- ‘for a given enzyme’, rephrase to make this more explicit and explain again why the 4 enzymes under study are so interesting
A sentence clearly stating which enzymes were used in this study and why they are interesting in understanding the N-glycan maturation pathway was added.

Page 13, section starts with ‘We used MD simulations’
- sentence ‘An effective enzyme accessibility…binding modes.’ is too long and unclear, please rephrase for readability
We split this long sentence into two and re-phrased it for more clarity.

- ‘when binding to a-mannosidase’, can the authors specify which mannosidase?
We specified which mannosidases are meant (mannosidase from the GH family 47).

Page 14, top section
- ‘structural differences of ER Man 1 and GnT 1’ define these differences, or link to a suitable reference
Two references describing the crystal structure of ER Man I and GnT I, respectively, were added.

Page 14, section start with ‘We perturbed’
- ‘We perturbed glycan-protein interactions…’ I don’t think the data convincingly shows that the interactions were indeed perturbed by reduction and alkylation, or at least the authors do not give a lot of proof here. I suggest rephrasing this to better reflect the small changes observed in kinetics of the alkylated proteins, as the authors also do a few sentences later.
We agree that perturbed is misleading in this context and adapted the sentence accordingly.
- ‘Similarly’ is in my opinion the wrong connector, as denaturing is not similar to alkylation.
We removed the word “similarly” and adapted the sentence.


Figure 1C. y axis should be ratio. the bar called ‘any’ is confusing.
We adapted the label of the y axis accordingly. Further we clarify the meaning and the relevance of the ‘any’ bar on page 7 again.

Legend Figure 2B: It is confusing what is plotted here, is it conversion/consumption of starting material Man9GlcNAc2, or production of product Man5GlcNAc2, as the y-axis suggests? Are there any intermediates observed, or is it a clean conversion from Man9 to Man5? Same question holds for Figure 2D.
What is shown in this graph is the generation of the final product (Man5GlcNAc2). Intermediates are produced by both enzymes GM I (2B) and GM II (2D). We study the appearance and disappearance of these intermediates and the kinetics of these events in more detail in figure 4 and the section “Rate constants describing appearance and disappearance of intermediate structures”. However we thank the reviewer for this comment and agree this should have been explained more carefully. We therefore added a sentence to the figure legend of 2B and 2D to clarify what is shown here and also refer the reader to figures 4A and B for more information about intermediate structures.

Figure 5B. I suggest to use different tick symbols, as it is quite difficult to distinguish between native and red/alkyl. Or make clear which one is dashed and solid lines.
We adapted the symbol of the red/alkyl results. Further the legend also contains the information on the line style now.

08-Apr-2021

Dear Prof Aebi:

Manuscript ID: CB-ART-01-2021-000019.R1
TITLE: Glycan-Protein Interactions Determine Kinetics of N-Glycan Remodeling

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after the minor revisions suggested.

Please revise your manuscript to fully address the reviewers’ comments. 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 in 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

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

Reviewer 1

The authors have addressed all my concerns, and therefore I support the publication of the manuscript. As a suggestion, the authors should use a "line" representation without hydrogen atoms instead of sticks for the glycans shown in Figures S2 and S3.

Reviewer 2

In my opinion, the revised version of the manuscript by Mathew et al can be acceptor for publication in RSC Chemical Biology. It is great to see that the authors did such a careful job addressing all comments, which in my opinion greatly improved the readability of the manuscript. It is an exciting study, and I want to congratulate the authors with the resulting manuscript.

One comment:
p.7 almost at the bottom: "Sites 2 and 5 tended to expose individual while" is missing a word after individual

We are pleased to read that both reviewers are mostly satisfied with the previously revised version of our manuscript and would like to thank the reviewers again for their very valuable input. We would like to mention that we added a small note on the choice of molecular force fields and correspondingly important citations for proper scientific correctness and recognition (page 17, Material and Methods).

Referee: 1

Comments to the Author
The authors have addressed all my concerns, and therefore I support the publication of the manuscript. As a suggestion, the authors should use a "line" representation without hydrogen atoms instead of sticks for the glycans shown in Figures S2 and S3.

We thank the reviewer for his/her suggestion, and we tested the representation of the glycans as lines. Unfortunately, the line representation lacks the three-dimensional impression due to the loss of shadings. Thus, we prefer to keep the stick representation of the glycans.

Referee: 2

Comments to the Author
In my opinion, the revised version of the manuscript by Mathew et al can be acceptor for publication in RSC Chemical Biology. It is great to see that the authors did such a careful job addressing all comments, which in my opinion greatly improved the readability of the manuscript. It is an exciting study, and I want to congratulate the authors with the resulting manuscript.

One comment:
p.7 almost at the bottom: "Sites 2 and 5 tended to expose individual while" is missing a word after individual

We thank the reviewer for his/her great eye for detail, the missing word is “branches” and we added it to the sentence.

13-Apr-2021

Dear Prof Aebi:

Manuscript ID: CB-ART-01-2021-000019.R2
TITLE: Glycan-Protein Interactions Determine Kinetics of N-Glycan Remodeling

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.

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