From the journal RSC Chemical Biology Peer review history

11-Jan-2021

Dear Dr Julian:

Manuscript ID: CB-ART-12-2020-000224
TITLE: A two-trick pony: lysosomal protease cathepsin B possesses surprising ligase activity

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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Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

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

Reviewer 1

The authors report an interesting finding where Cathepsin B goes beyond its protease activity to serve as a ligase. Here, Julian and co-workers demonstrated the combination of protease-ligase activity with a set of peptides. Further, they investigated the sequence specificity, activity with L-isoAsp isomer, along with other control experiments. At this point, I was expecting to see how the potential of this tool is validated. The authors should consider including at least one application to demonstrate the same. Also, the authors should include LC-MS data for all the experiments in the supplementary information.

Reviewer 2

Julian and coworkers detected in the cysteine protease cathepsin B a ligation activity. Using a set of model peptides they found a dipeptide ligation activity that results in the cleavage and fragment religation of model peptides. They were also able to show that a recognition peptide and dipeptide can be ligated albeit with relatively low yields. They also show that by utilization of a peptide that contains L-isoAsp at position two thus mimicking a dipeptide sequence, ligation can be observed. The activity requires (as one would expect) the presence of the active site cys. Experiments are sound and these finding ad a new function to cathepsin B which is an important enzyme in various cellular processes.
However both in the abstract and in the discussion the authors give the impression that this enzyme can be used for general purpose peptide ligation. For example, they state: „this method of utilizing L-isoAsp to ligate longer sequences could be used to produce longer canonical peptide sequence” In view of the extremely low yield found and documented in Figure 6c this is more wishful thinking rather than reality or being close to reality. Moreover, the authors completely ignore the existence of proteases that were engineered into ligases. The famous example of Jim Wells for converting subtilisin into subtiligase by converting the enzyme from a serin protease into a cysteine protease (as cathepsin B is) remained unmentioned. See https://pubs.acs.org/doi/10.1021/acs.chemrev.9b00372 The same holds true for trypsilgase. Both subtiligase and trypsiligase require a peptide carboxylester substrate but result in traceless ligation in high yields and in contrast to the (future) use of cathepsin B (mutants) with isoAsp peptide substrate their use does do not require further downstream enzymatic modifications. The authors only indirectly mention butelase as a ligation enzyme which has also been established for broader applications in peptide ligation.
In conclusion, the authors should more focus on the careful characterization of the cathespsin B ligation activity rather than advertising this activity as an unmet need in chemical peptide and protein synthesis.

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

We have carefully revised our manuscript in accordance with the reviewer suggestions. We have added a section to the introduction with references to place our work in better context. We have also clarified that this report focuses on reporting novel and interesting ligation rather than development of a tool. The additional data requested has also been added to the supporting information. We feel that the manuscript is now much improved and ready for publication.

A detailed response is attached.

We thank the reviewers for their comments that have helped us improve our manuscript.
Referee: 1 The authors report an interesting finding where Cathepsin B goes beyond its protease activity to serve as a ligase. Here, Julian and co-workers demonstrated the combination of protease-ligase activity with a set of peptides. Further, they investigated the sequence specificity, activity with LisoAsp isomer, along with other control experiments. At this point, I was expecting to see how the potential of this tool is validated. The authors should consider including at least one application to demonstrate the same.
Our primary goal in this work is to draw attention to and characterize the previously unreported ligase ability of catB. To clarify this, the text has been rewritten in the context of currently existing dual-activity enzymes and the potential future optimizations and applications of catB.
Also, the authors should include LC-MS data for all the experiments in the supplementary information.
All LC-MS data has been included in the main text of the manuscript or in the SI.
Referee: 2 Julian and coworkers detected in the cysteine protease cathepsin B a ligation activity. Using a set of model peptides they found a dipeptide ligation activity that results in the cleavage and fragment religation of model peptides. They were also able to show that a recognition peptide and dipeptide can be ligated albeit with relatively low yields. They also show that by utilization of a peptide that contains L-isoAsp at position two thus mimicking a dipeptide sequence, ligation can be observed. The activity requires (as one would expect) the presence of the active site cys. Experiments are sound and these finding ad a new function to cathepsin B which is an important enzyme in various cellular processes. However both in the abstract and in the discussion the authors give the impression that this enzyme can be used for general purpose peptide ligation. For example, they state: „this method of utilizing L-isoAsp to ligate longer sequences could be used to produce longer canonical peptide sequence” In view of the extremely low yield found and documented in Figure 6c this is more wishful thinking rather than reality or being close to reality. Moreover, the authors completely ignore the existence of proteases that were engineered into ligases. The famous example of Jim Wells for converting subtilisin into subtiligase by converting the enzyme from a serin protease into a cysteine protease (as cathepsin B is) remained unmentioned. See https://pubs.acs.org/doi/10.1021/acs.chemrev.9b00372 The same holds true for trypsilgase. Both subtiligase and trypsiligase require a peptide carboxylester substrate but result in traceless ligation in high yields and in contrast to the (future) use of cathepsin B (mutants) with isoAsp peptide substrate their use does do not require further downstream enzymatic modifications. The authors only indirectly mention butelase as a ligation enzyme which has also been established for broader applications in peptide ligation. In conclusion, the authors should more focus on the careful characterization of the cathespsin B ligation activity rather than advertising this activity as an unmet need in chemical peptide and protein synthesis.
We thank the reviewer for directing our attention to additional protease/ligases that we overlooked. We have added a paragraph to the intro discussing dual-activity enzymes in greater detail, including some of RSC Chemical Biology Page 2 of 37 the methods used to increase yield, and their applications. Potential steps for optimization and possible applications are also discussed in the body of text and the conclusion in the context of other current ligases and how catB may serve as a complement by introducing new reaction possibilities.
Citations added for currently existing protease/ligases and their optimization history-
• Lewinska, M.; Seitz, C.; Skerra, A.; Schmidtchen, F. P. A Novel Method for the N-Terminal Modification of Native Proteins. Bioconjug. Chem. 2004, 15 (2), 231–234.
• Chang, T.K.; Jackson, D.Y.; Burnier, J.P.; Wells, J.A. Subtiligase: A tool for semisynthesis of proteins. PNAS. 1994, 91, 12544-12548.
• Liebscher, S.; Schöpfel, M.; Aumüller, T.; Sharkhuukhen, A.; Pech, A.; Höss, E.; Parthier, C.; Jahreis, G.; Stubbs, M.T.; Bordusa, F. N-terminal protein modification by substrate activated reverse proteolysis. Angew. Chem. Int. Ed. 2014, 53(11), 3024-3028.
• Nguyen, G.K.T.; Hemu, X.; Quek, J.P.; Tam, J.P. Butelase-Mediated Macrocyclization of D-AminoAcid-Containing Peptides. Angew. Chem. Int. Ed. 2016, 55, 12802-12806.
Citation added for optimization of reaction yield-
• Tan, X.; Yang, R.; Liu, C.F. Facilitating Subtiligase-Catalyzed Peptide Ligation Reactions using Peptide Thioester Substrates. Org. Lett. 2018, 20, 6691-6694.
Citations added for applications of protease/ligase enzymes-
• Wildes, D. and Wells, J.A. Sampling the N-terminal proteome of human blood. PNAS. 2010, 107(10), 4561-4566.
• Nguyen, G.K.T.; Cao, Y.; Wang, W.; Fa Liu, C.; Tam, J.P. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide. Angew. Chem. Int. Ed. 2015, 127, 15920-15924.
We also agree that this report does not provide an optimized tool and have made sure that the text make this point more clear. The conclusion now states, “Future experiments could be performed to optimize the ligase yield of catB by mutagenesis of the residues in close proximity to the active-site, as in the case of previously engineered enzymes, or through the use of modified peptide substrates.”

10-Feb-2021

Dear Dr Julian:

Manuscript ID: CB-ART-12-2020-000224.R1
TITLE: A two-trick pony: lysosomal protease cathepsin B possesses surprising ligase activity

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,

Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

Reviewer 2

The authors addressed the critical points that were raised by the reviewers in this revised manuscript. It can now be recommended for publication.
Typo P3: A trypsin variat should read variant.