Identification and Characterization of ShSPI, a Kazal-Type Elastase Inhibitor from the Venom of Scolopendra Hainanum

Round 1

Reviewer 1 Report

Identification and characterization of ShSPI, a kazal type elastase inhibitor from the venom of Scolopendra hainanum

In the present study, the researchers have identified a kazal-type serine protease inhibitor, ShSPI from the venom of Scolopendra hainanum (centipedes). They compared ShSPI with the current pharmaceutical compound Sivelestat. They demonstrate that ShSPI showed significant inhibitory effects on porcine pancreatic elastase (Ki = 225.83 nM) and human neutrophils elastase (Ki = 12.61 nM). They propose that ShSPI may have several important physiological and clinical applications such as pharmaceutical options for the treatment of protease-related cardiopulmonary diseases.

Overall, I believe the identification of the novel kazal-type elastase inhibitor (ShSPI) from the venom of centipedes is interesting and its application in comparison to the existing SPI inhibitor compound Sivelestat will be useful from a physiological perspective for clinical researchers and enzymologists.

Comments:

In Figure 3, it is important that the authors used the human neutrophil elastase (HNE) as a target for ShSPI inhibition in comparison with the pharmaceutical Sivelestat. However, the authors used the pancreatic version of a porcine elastase as a comparison between previous studies with Sivelestat and PPE.

 

What is the sequence similarity between PPE and the human variant of PPE and HNE? It would be useful in this figure if the authors presented the sequences of the PPE, HNE and the human variant of PPE.

 

It would also be helpful if the authors depicted the proposed interaction site of ShSPI to HNE. A binding/interaction comparison between ShSPI and Sivelestat to the elastase would be impactful to highlight the potential clinical use of ShSPI.

 

In Figure 1, the authors refer to Schechter and Berger for the nomenclature of the important ShSPI sites. This nomenclature is described based on the cleavage site of the substrate using an appropriate enzyme.

Which enzyme is the reference enzyme, elastase (PPE, HNE)?

Is ShSPI being cleaved? if so, what is the proof of this? How is it acting to inhibit elastase (Kazal 1 / Kazal 2 / other mechanism?

 

While the structural similarities were compared between inhibitors, Overall, more information in the manuscript (discussion) is required on how this inhibitor may be functioning as a kazal-type elastase inhibitor (atypical) with other known SPIs in its class.

 

In Table 3, the authors took an alanine mutagenesis approach to determine which sites in ShSPI were important for protease inhibition. There was a wide range of mutations all along the peptide that altered the function of the inhibitor. What information based on the structure and sequence of the inhibitor does this tell us about the protein? This has not been made clear in the paper and more rationale would be appreciated surrounding this experiment and results.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled "Identification and characterization of ShSPI, a kazal-type elastase inhibitor from the venom of Scolopendra hainanum" describes the identification, synthesis, refolding, structure elucidation, biological evaluation and plasma stability of the novel elastase inhibitor ShSPI produced by the centipede species Scolopendra hainanum. Also, the amino acid residues playing a critical role in the structure/activity of this peptide are experimentally identified. As the author indicated, elastase inhibitors could be excellent candidates for the treatment of several diseases generated by the uncontrolled activity of human neutrophil elastase.
The content of the manuscript is within the scope of the journal; it is concise, organized, and should be interesting for a large number of readers. However, the manuscript lacks information in the Materials and Methods section since few details are provided at least on chromatography, mass spectrometry, and DNA sequencing experiments. Also, the introduction lacks information about elastase inhibitors found in other venomous species; therefore the reader may perceive the idea that only a few elastase inhibitors are known. If this is the case, the author should clearly state it or mention elastase inhibitors found at least in other venomous species, including centipedes.
Additionally, I would like to suggest some corrections:
Line 51: was instead of "were", since it refers to "a protein precursor."
Line 55: using the term "homology" is not adequate, replace with "sequence similarity."
Figure 1. The percentage of sequence similarity should be shown for every sequence to make clear how similar they are in comparison with ShSPI.
Line 77. Refolding instead of "the refolding". Linear instead of "Linner". Refolding analysis was performed instead of "refolding was performed..."
Line 80. were formed instead of "were refolded."
Line 88. It has been reported instead of "It has reported".
Line 90. illustrate instead of "illustrated"
Line 94. respectively instead of "repectively"
Line 190. were synthetized instead of "are synthetized"
Line195. ..these mutants are listed instead of "mutants were listed"
Line 203. I guess the authors meant "physiological condition" instead of "physical condition"
Line 228. has a stable instead of "had a stable"
Line 230. higher selectivity instead of "more selectively"
Line 233. "In consistence with" would fit better.
Line 233. ShSPI specifically instead of "ShSPI could specifically".
Line 260. MALDI-TOF?

Author Response

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Reviewer 3 Report

The authors found the kazal-type elastase inhibitor ShSPI from centipede venom. Using chemically synthesized ShSPI, they examined the inhibitory activity of ShSPI, identified amino acid residues essential for activity, and determined its three-dimensional structure.
Major points
1. How did the authors find ShSPI cDNA? Was ShSPI cDNA the main component of centipede venom cDNA, or was one of the sequenced cDNAs happened to be ShSPI?
2. It has not been confirmed that ShSPI is actually present in centipede venom. It is better to examine elastase inhibitory activity in centipede venom.
3. The authors must explain why ShSPI does not include an N-terminal QRNRR.
Minor points
1. line 94, 50 nM → 500 nM
2. line 203, physical → physiological?

Author Response

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Reviewer 4 Report

After careful revision of the manuscript it was found that the manuscript covers an important topic and worthy of study and can be published after minor revision which does not affect the quality of the manuscript. I believe that some references could enrich this manuscript, for instance:

 

SPIXIANA   Vol. 35, 19-27 (2012).

TOXICON   Vol. 143,  96-107 (2018).

JOURNAL OF CELLULAR BIOCHEMISTRY   Vol. 120, 14594-14603 (2019).

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

No additional comments.