From the journal RSC Chemical Biology Peer review history

03-Feb-2022

Dear Dr Jackson:

Manuscript ID: CB-ART-12-2021-000239
TITLE: Computational design and experimental characterisation of a stable human heparanase variant

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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Yours sincerely,
Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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Reviewer 1

This is a very nice paper, showing the possibility to substantially stabilise an important enzyme via rational design. The authors introduce rationally 26 mutations, which allow them to crystallise heparanase.
I found the paper very interesting, well written and of potential interest to a number of researchers. It is one of the rare cases where the paper should be accepted as it is.

Reviewer 2

In this work, Whitefield et al. study the structure and dynamics of heparinase. The authors stabilized heparinase by using an online tool developed by Fleishmann lab (PROSS), then they solved the structure and run the molecular dynamics of the variants to understand the effect of the stabilizing mutations on the overall structure. Despite 26 mutations, heparinase maintains identical kinetics, inhibition, structure and protein dynamics. The scope of PROSS is enhancing stability and expression without changing protein function, however it has been often reported an increase of activity due to the trade-off stability/activity. This work represents a nice exception where stability increase without increase in activity. The authors study in details structure and dynamics to explain their results. The paper is robust, well written and conclusions are supported by the data.

Minor comments/corrections:

1. In mammalian cells, it expresses with glycosylation at six sites, according to figure no 1. This glycosylation might be necessary for function. In bacterial systems, a protein expressed without glycosylation. Will it affect the enzyme function? Can the author comment on this?
2. The authors mention that size of the heparinase is 50 kDa, bit later in the text as 44kDa. Is the glycosylation at six sites count for 6kDa? It is unclear.
3. I am also confused by the small subunit that is mentioned as 8kDa in the introduction but then reported as 10kDa in the caption fig2.
4. Table 1. Please consistent with name WT HPSE/HPSE We, HPSE P6/ P6 HPSE. Same for the mutant name, either used single letter or one letter throughout the text including figure.
5. Page number 3, left column: which are the five mutations mentioned here?
6. Figure 1: typo Mamalian to Mammalian

We thank the reviewers for the comments to this manuscript and we have made all recommended changes and responded to the comments below. Thank you for your time to review this manuscript.

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

Referee: 1
Comments to the Author
This is a very nice paper, showing the possibility to substantially stabilise an important enzyme via rational design. The authors introduce rationally 26 mutations, which allow them to crystallise heparanase.

I found the paper very interesting, well written and of potential interest to a number of researchers. It is one of the rare cases where the paper should be accepted as it is.

Referee: 2
Comments to the Author
In this work, Whitefield et al. study the structure and dynamics of heparinase. The authors stabilized heparinase by using an online tool developed by Fleishmann lab (PROSS), then they solved the structure and run the molecular dynamics of the variants to understand the effect of the stabilizing mutations on the overall structure. Despite 26 mutations, heparinase maintains identical kinetics, inhibition, structure and protein dynamics. The scope of PROSS is enhancing stability and expression without changing protein function, however it has been often reported an increase of activity due to the trade-off stability/activity. This work represents a nice exception where stability increase without increase in activity. The authors study in details structure and dynamics to explain their results. The paper is robust, well written and conclusions are supported by the data.

Minor comments/corrections:
1. In mammalian cells, it expresses with glycosylation at six sites, according to figure no 1. This glycosylation might be necessary for function. In bacterial systems, a protein expressed without glycosylation. Will it affect the enzyme function? Can the author comment on this?

Comment added to the manuscript, page 2 paragraph 3 and page 3 paragraph 1. “The loss of the six glycosylation sites no effect on the activity of the enzyme, suggesting these sites may be important for protein solubility in mammalian systems.”

2. The authors mention that size of the heparinase is 50 kDa, bit later in the text as 44kDa. Is the glycosylation at six sites count for 6kDa? It is unclear.

This is mentioned in caption to Figure 2 (a) “noting that the size of the large subunit is smaller than the previously reported value of 50 kDa due to the lack of glycosylation”

3. I am also confused by the small subunit that is mentioned as 8kDa in the introduction but then reported as 10kDa in the caption fig2.

Updated to 8 kDa within the caption fig2

4. Table 1. Please consistent with name WT HPSE/HPSE We, HPSE P6/ P6 HPSE. Same for the mutant name, either used single letter or one letter throughout the text including figure.

Updated title on table 1 with HPSE P6/WT

5. Page number 3, left column: which are the five mutations mentioned here?

Updated on page 3 paragraph 2 to “To understand how the 26 mutations in HSPE P6 result in enhanced protein folding and stability” instead of 5 mutations.

6. Figure 1: typo Mamalian to Mammalian

Updated in figure 1.

11-Feb-2022

Dear Dr Jackson:

Manuscript ID: CB-ART-12-2021-000239.R1
TITLE: Computational design and experimental characterisation of a stable human heparanase variant

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form.

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With best wishes,

Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology