From the journal RSC Chemical Biology Peer review history
Bioorthogonal protein labelling enables the study of antigen processing of citrullinated and carbamylated auto-antigens
Round 1
Manuscript submitted on 13 Қаң. 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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19-Feb-2021
Dear Prof van Kasteren; dear Sander:
Manuscript ID: CB-ART-01-2021-000009
TITLE: Bioorthogonal Protein Labelling Enables The Study Of Antigen Processing of Citrullinated and Carbamylated Auto-Antigens
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.
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I look forward to receiving your revised manuscript.
Yours sincerely,
Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
************
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Reviewer 1
This manuscript by van Kasteren and coworkers has been considerably improved by implementing the suggested reviewer's comments.
Nonetheless, some minor issues to fix:
- Abstract: imaged the of the commonly - truncated sentence
Introduction
- Page 6: "this is none the more urgent" - rephrase for clarity
- Right column "its cleavage by asparagine endopeptidase leads*" - leading
- Page 7: "as long as their fold remains intact. The latter" - rephrase for clarity
Results and Discussion
- Page 8: m.w. should be M.W.
- Page 9: this insinuates*? - suggests
With these modifications, the paper can be accepted for publication at RSC Chem Bio.
Reviewer 2
This manuscript composed by van Kasteren, Toes and coworkers describes their studies towards the application of CuAAC bioconjugation chemistry for proteolysis of recombinant proteins carrying metabolic incorporated non-natural amino acid residues as trackable antigens. With the bioorthogonal functional groups in present, the antigen process and presentation at the initial stage of the adaptive immune response could be monitored at any staged if necessary. The authors demonstrated the irreplaceable value of click chemistry in proteolytic analysis through detailed experiments and in-depth research via microscopic, mass spectroscopic studies and in-gel fluorescence presentations. Despite the great efforts spending on comparison of a variety of biochemical labelling methods, it is still difficult to track the modification process of these antigen in real time, achieving the original concept of a bioorthogonal protein labelling. Considering the particularity of the protein environment in the endo-lysosomal system of dendritic cells, it seems that there is an urgent demand for specialized optimization of bioorthogonal labelling reagents toward these antigens. In my opinion, the novelty and innovation of this research work build a solid foundation for further studies, making it suitable to be published in the RSC Chemical Biology.
Minor point:
The background reactivity of the other available bioorthogonal chemistries might be caused by their high reactivity toward the intracellular remnant of Aha or Hpg or off-target proteins, especially when metabolic incorporation of the UAA is the scenarios. Is there any other explanation if it is only evaluated by fluorescence imaging?
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This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters
Dear Dr Bernardes, Dear Gonçalo,
We are very happy with the positive evaluations of the two reviewers and would like to thank them for these supporting words. We have addressed all their issues in a point-by-point manner in the attached document.
Kind regards,
Sander van Kasteren and René Toes
Reviewer 1:
Nonetheless, some minor issues to fix:
- Abstract: imaged the of the commonly - truncated sentence Introduction
This has been changed to ‘imaged the post-uptake fate of the commonly’
- Page 6: "this is none the more urgent" - rephrase for clarity
This has been rephrased to: ‘this is particularly important during antigen processing”
- Right column "its cleavage by asparagine endopeptidase leads*" – leading
‘Leads’ has been changed to ‘leading’
- Page 7: "as long as their fold remains intact. The latter" - rephrase for clarity Results and Discussion
This paragraph has been simplified to read:
So far, the go-to approach for studying antigen-routing has been the use of detectable model proteins
(e.g. lactamases27, luciferase28, and HRP29) or fluorophore-modified model antigens30-32. Reporter
proteins are only active (and thefore detectable) as long as their structure . Fluorescent modification of
antigens changes the charge, hydrophobicity/aromaticity, shape and structure, which alters the
properties of the protein.33, 34 We hypothesize that this leads to significant structural alterations that
strongly affect processing and antigenicity as shown for the examples above.
- Page 8: m.w. should be M.W.
Corrected
- Page 9: this insinuates*? – suggests
‘Insinuates’ has been changed to ‘suggests’
Reviewer 2:
The background reactivity of the other available bioorthogonal chemistries might be caused by their
high reactivity toward the intracellular remnant of Aha or Hpg or off-target proteins, especially when
metabolic incorporation of the UAA is the scenarios. Is there any other explanation if it is only
evaluated by fluorescence imaging?
This is a very interesting point. Fluorescence imaging alone greatly complicates the evaluation of UAAcontaining proteins (and any other compounds for that matter). Hence we have corroborated everything by gel imaging. If the Aha and Hpg do react in the antigen presenting cell, one could indeed
envisage the formation of reactive products that can give other background reactions. Especially
partially oxidized variants of the alkyne and the azide-groups could give background reactions.
However, in our previous work (Bakkum et al. ACS Chem Biol 2018), showed that azides and alkynes
are remarkably stable even to the conditions found in the dendritic cell endo-lysosomal compartment.
It does raise a great set of experiments, where we can use cleavable linkers and the beads from the
aforementioned paper to analyse the chemical degradation and by-products of commonly used click
reactions and determine the extent of ‘true’ bioorthogonality.
Round 2
Revised manuscript submitted on 22 Ақп. 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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22-Feb-2021
Dear Dr van Kasteren:
Manuscript ID: CB-ART-01-2021-000009.R1
TITLE: Bioorthogonal Protein Labelling Enables The Study Of Antigen Processing of Citrullinated and Carbamylated Auto-Antigens
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.
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With best wishes,
Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
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