Primary Structure and Conformation of a Tetrodotoxin-Binding Protein in the Hemolymph of Non-Toxic Shore Crab Hemigrapsus sanguineus

Round 1

Reviewer 1 Report

The manuscript entitled "Primary structure and conformation of a tetrodotoxin-binding protein in the hemolymph of non-toxic shore crab Hemigrapsus sanguineus"by Yuji Nagashima et al., reports the TTX-binding proteins isolated from non-toxic shore crab and elucidates the primary structure and charactetistics. The topics in this manuscript are interesting and these results will provide possible reference for antidotes for TTX intoxication. However, the analysis in the anuscript has some problems.

I think the rpm should be same between Line 106(18,800g)  and Line 124 (18,000g, not 18000g).
Line 124, please provide the concentration of precipitate dissolved in NaCl- sodium phosphate buffer.
Line 171, please provide  computational analysis softwares or methods for predicting key amino acids in TfVWF and docking analyses.
Line 272-277, for isolation of TTX-binding protein, fraction I and II  have only one band and  are homogeneity, why do they have different retention times and binding activity.
Line 291, HSTPB should be a trimerical conformation.
Line 297, please provide a clearer SDS-PAGE image of figure 2-D.
Line 417-418, authors should provide evidences for determining that the subunits arebound by disulfide bonds.
Line 428-429, what do the authors think about the molecular masses difference between previous and present studes.

Author Response

Response to the reviewer’s comments (Reviewer 1)

 

Thank you for your considerable review and helpful suggestion. According to your suggestion, the manuscript has been revised.

 

• I think the rpm should be same between Line 106 (18,800g) and Line 124 (18,000g, not 18000g).

 

Response: The rpm was revised to 18,800g in line 125, page 3 of the revised manuscript.

2) Line 124, please provide the concentration of precipitate dissolved in NaCl-sodium phosphate buffer.

 

Response: The protein amount of the precipitate is 3,970 mg. The score is added in line 125, page 3 of the revised manuscript.

 

• Line 171, please provide computational analysis softwares or methods for predicting key amino acids in TfVWF and docking analyses.

 

Response: We did not predict key amino acids in TfVWF and docking analysis in this study. If the reviewer would like to know them, please see the reference [18] of the revised manuscript.

 

4) Line 272-277, for isolation of TTX-binding protein, fraction I and II have only one band and are homogeneity, why do they have different retention times and binding activity.

 

Response: I think that the amino acid composition of fraction I and fraction II may be slightly different from each other. Therefore, they may have different retention times and binding activity.

5) Line 291, HSTPB should be a trimerical conformation.

 

Response: The ratio of molecular mass of the intact HSTBP to that of the sum of the three subunits is 2.37. I think, therefore, HSTBP is likely to be a dimeric conformation.

 

6) Line 297, please provide a clearer SDS-PAGE image of figure 2-D.

 

Response: Unfortunately, we have only the SDS-PAGE shown in Fig 2-D.

 

7) Line 417-418, authors should provide evidences for determining that the subunits are bound by disulfide bonds.

 

Response: Reduction with dithiothreitol resulted in cleave off HSTBP to three subunits. Please see line 413, page 14 of the revised manuscript.

 

8) Line 428-429, what do the authors think about the molecular masses difference between previous and present studies.

 

Response: Molecular weight measurements by electrophoresis analysis are not as accurate as those by mass spectrometry. Therefore, results may vary depending on the analysis methods used, such as gels and markers.

Author Response File: Author Response.pdf

Reviewer 2 Report

Toxins of different origins, including tetrodotoxin (TTX), quite often act as potent neurotoxins. Originally they were recognized as poisons for their lethal effects on humans. Nonetheless, we have recently learned that when administered at doses far below LD50 they might display/exhibit therapeutic properties to treat pain originating from different kinds of diseases, including cancer. In their manuscript titled "Primary structure and conformation of a tetrodotoxin-binding protein in the hemolymph of non-toxic shore crab Hemigrapsus sanguineus" Nagashima Y. and colleagues report the identification of a TTX-binding protein from the non-toxic marine shore crab Hemigrapsus sanguineus (HSTBP). Starting with a biochemical approach they first purified and isolate the HSTBP. Afterward, they characterized the complex identifying three different subunits one of which (subunit 2) appears to be N-glycosylated. Eventually, with a reverse genetic approach, they cloned the gene encoding for the HSTBP, and with the help of a recombinant protein (subunit 2) they assessed the binding activity to TTX. Overall, I appreciated the manuscript. However, prior to publication, there are a couple of minor issues that need to be sorted out, and those are below shortly summarized. Minor concerns

1. Throughout the main text I noticed a couple of typos (e.g. line 62: I guess principle instead "...pinciple..."; line 211: after colon lower case instead of capital letter).
2. A couple of inaccuracies should be edited (e.g. line 141 I guess that the authors refer to a gradient gel when they state "...3-10 polyacrylamide gel...")
3. Figure 2D: the authors should clarify the meaning of using transferrin, I guess that it works as a control because of N-glycosylated. Nonetheless, it should be made explicit. Furthermore, in the lane "Sample +" there is an extra band at around 30 kDa. What is that? It should be discussed. Finally, the authors should clarify whether the HSTBP is exclusively N-linked glycosylated or putative O-linked glycosylation might occur. In the latter case treatment with alkali, for example hydrazinolysis, or other O-glycopeptidases are suggested.
4. To the reviewer, it is not clear to what database refers the accession number LC733238 (line 324). It should be clarified.
5. Line 341: it is not clear whether the pI refers to the supramolecular complex or to a specific subunit. In the latter case, to which one?
6. Lines 342-347: it is not clear whether the % refers to similarity or identity.
7. Figure 4: from the alignment is quite hard to infer the relevance of Cysteine (C) residues. The alignment shows that there are also other residues highly conserved among the different species, including W, G, and E, but only C is highlighted. Later in the section "Conclusions" the authors shortly discuss that C might be involved in the formation of disulfide bonds. This issue should be already shortly discussed in the "Results" section and shortly substantiated in the figure legend.
8. Line 389: what is the unit of measurement of the figure in brackets? Please provide it.
9. To the reviewer is not clear how the protein is cleaved into three different peptides. Are there any potential cleavage recognition sequences for protease/s, or chemical/s? This issue needs more attention throughout the manuscript and should be articulated.

 

Author Response

Response to the reviewer’s comments (Reviewer 2)

 

Thank you for your considerable review and helpful suggestion. According to your suggestion, the manuscript has been revised.

 

• Throughout the main text I noticed a couple of typos (e.g. line 62: I guess principle instead "...pinciple..."; line 211: after colon lower case instead of capital letter).

 

Response: “pinciple” is a typo for “principle” in line 63, page 2 of the revised manuscript.

Capital letter after colon was revised to lowercase in line 211, page 5 of the revised manuscript.

 

• A couple of inaccuracies should be edited (e.g. line 141 I guess that the authors refer to a gradient gel when they state "...3-10 polyacrylamide gel...")

 

Response: “3-10” was deleted in line 142, page 3 of the revised manuscript.

 

• Figure 2D: the authors should clarify the meaning of using transferrin, I guess that it works as a control because of N-glycosylated. Nonetheless, it should be made explicit. Furthermore, in the lane "Sample +" there is an extra band at around 30 kDa. What is that? It should be discussed. Finally, the authors should clarify whether the HSTBP is exclusively N-linked glycosylated or putative O-linked glycosylation might occur. In the latter case treatment with alkali, for example hydrazinolysis, or other O-glycopeptidases are suggested.

 

Response: Transferrin was used as a control of N-glycosylated protein. Therefore, the sentence “Transferrin was used as a control of N-glycosylated protein” was added to the legends for Figure 2 in lines 306-307, page 8 of the revised manuscript.

HSTPB is likely to be N-linked glycosylated because it was retained on the lectin affinity Con A-Sepharose column and eluted with an eluent containing methyl-alpha-D-mannopyranoside.

 

• To the reviewer, it is not clear to what database refers the accession number LC733238 (line 324). It should be clarified.

 

Response: Database is closed to the public until the paper is published.

 

• Line 341: it is not clear whether the pI refers to the supramolecular complex or to a specific subunit. In the latter case, to which one?

 

Response: pI is to HSTBP, not subunits. The sentence of pI was revised to “The isoelectric point (pI) of HSTBP was estimated to be 5.26 using DANASIS pro.” in line 339, page 9 of the revised manuscript.

 

• Lines 342-347: it is not clear whether the % refers to similarity or identity.

 

Response: The % refers to identity. The word “similarity” was changed to “identity” in line 340, page 9 of the revised manuscript.

 

• Figure 4: from the alignment is quite hard to infer the relevance of Cysteine (C) residues. The alignment shows that there are also other residues highly conserved among the different species, including W, G, and E, but only C is highlighted. Later in the section "Conclusions" the authors shortly discuss that C might be involved in the formation of disulfide bonds. This issue should be already shortly discussed in the "Results" section and shortly substantiated in the figure legend.

 

Response: Cystein (C) is highlighted because the subunits is suggested to be disulfide bonded by SDS-PAGE.

 

• Line 389: what is the unit of measurement of the figure in brackets? Please provide it.

 

Response: The unit is Da. The words “TTX (319)” was revised to “TTX (319 Da)” in line 386, page 13 of the revised manuscript.

 

• To the reviewer is not clear how the protein is cleaved into three different peptides. Are there any potential cleavage recognition sequences for protease/s, or chemical/s? This issue needs more attention throughout the manuscript and should be articulated.

 

Response: Thank you for your suggestion. At present, we are not sure the mechanism of cleave off.

Author Response File: Author Response.pdf

Reviewer 3 Report

The toxic effects of TTX,  a potent neurotoxin, are highly specific to certain animals making it a useful tool for studying the molecular basis of toxicity and the mechanisms of action of toxins in general. In this manuscript, Nagashima et al. have isolated and characterized a tetrodotoxin-binding protein (HSTBP) from the nontoxic marine shore crab.  The findings of this study provide insight into the presence and potential antitoxic function of tetrodotoxin-binding proteins in nontoxic animals. This is an interesting topic, and the authors did well on their experimental design and result presentation. The manuscript is generally well-written with informative figures. In general, I evaluate this as a good paper. I only have some minor revision suggestions. Their intention is to help the authors improve the manuscript further. They can be addressed through in-text revisions.

 

Line 19, The author should annotate the abbreviation "HSTBP".

 

Line 47, " one mouse unit (MU) is defined as …" The definition of MU should be moved forward.

 

Line 72, " HMWS was purified from the muscle ..". This sentence is lengthy. The author may consider revising to " HMWS with a molecular mass of 434 kDa, including two subunits, 272 and 47, was purified from the muscle of the …".

 

Line 79, Please add a ref after "… despite being non-toxic ".

 

Line 82, Consider revising to " In contrast, intraperitoneal or intravenous injection of hemolymph into mice prior to TTX injection effectively protected them from the lethal activity of TTX. ".

 

Line 104, The animal study was reviewed and approved by the institutional ethics committee.

 

Line 117, I think Table 1 should be moved to somewhere like line 264?

 

Line 119, "600 µg TTX was added to the pooled hemolymph sample (95 mL, 5300 mg protein)."

 

Line 124, 18,000g

 

Line 182, the version of Colabfold should be noted.

 

Line 189, Escherichia coli to E. coli.

 

Figure. 2. Please make sure that the resolution is ok in this Figure.

 

Line 390. Should be 5.51 µg

 

Line 474, 5,049 bp encoding 1,683. Please check this throughout the paper.

Line 471-484. This part is a little bit repetitive with the results and discussion. The author can focus on the major contribution of their findings (like potential antioxidants) and perspective in the future (like the binding mechanism of the TTX-binding protein HSTBP).

Comments for author File: Comments.pdf

Author Response

Response to the reviewer’s comments (Reviewer 3)

 

Thank you for your considerable review and helpful suggestion. According to your suggestion, the manuscript has been revised.

• Line 19, The author should annotate the abbreviation "HSTBP".

Response: According to the reviewer’s comment, the annotation of “HSTBP” was added in line 21, page 1 of the revised manuscript.

• Line 47, " one mouse unit (MU) is defined as …" The definition of MU should be moved forward.

Response: According to the reviewer’s comment, the definition of MU was moved to lines 45-47, page 1 of the revised manuscript.

• Line 72, " HMWS was purified from the muscle.". This sentence is lengthy. The author may consider revising to " HMWS with a molecular mass of 434 kDa, including two subunits, 272 and 47, was purified from the muscle of the …".

Response: According to the reviewer’s comment, the sentences were revised in lines 74-76, page 2 of the revised manuscript.

• Line 79, Please add a ref after "… despite being non-toxic ".

Response: A reference [21] was added after "… despite being non-toxic " in line 80, page 2 of the revised manuscript.

• Line 82, Consider revising to " In contrast, intraperitoneal or intravenous injection of hemolymph into mice prior to TTX injection effectively protected them from the lethal activity of TTX. ".

 Response: According to the reviewer’s comment, the sentences were revised in lines 83-84, page 2 of the revised manuscript.

• Line 104, The animal study was reviewed and approved by the institutional ethics committee.

 

Response: This study does not fall under animal experimentation ethics on mammals and reptiles, because crabs are crustaceans. We describe “Institutional Review Board Statement: Not applicable.” in line 481, page 15 of the revised manuscript.

• Line 117, I think Table 1 should be moved to somewhere like line 264?

Response: Table 1 shows details of the sample used in this study. Therefore Table 1 is shown in line 118, page 3 of the revised manuscript, according to advice by an English proofreader.  

• Line 119, "600 µg TTX was added to the pooled hemolymph sample (95 mL, 5,300 mg protein)."

Response: According to the reviewer’s comment, the sentences were revised in lines 120-121, page 3 of the revised manuscript

• Line 124, 18,000g,

Response: The rpm was revised to 18,800g in line 125, page 3 of the revised manuscript.

• Line 182, the version of Colabfold should be noted.

Response: The version (1.4.0) of Colabfold was noted in line 182, page 4 of the revised manuscript.

• Line 189, Escherichia coli to E. coli.

Response: According to the reviewer’s comment, the word was revised in line 189, page 4 of the revised manuscript.

• 2. Please make sure that the resolution is ok in this Figure.

Response: Unfortunately, we have only the SDS-PAGE shown in Fig 2.

• Line 390. Should be 5.51 µg

Response: I am afraid I didn't understand your comment.

• Line 474, 5,049 bp encoding 1,683. Please check this throughout the paper.

Response: We have checked.

• Line 471-484. This part is a little bit repetitive with the results and discussion. The author can focus on the major contribution of their findings (like potential antioxidants) and perspective in the future (like the binding mechanism of the TTX-binding protein HSTBP).

Response: Concluding remarks made concise as follows in lines 464-470, page 15 of the revised manuscript.:

This study revealed a novel TTX-binding protein, HSTBP, from the hemolymph of non-toxic shore crab H. sanguineus. The protein comprises three subunits, Arg³⁴-Gln²⁶¹ (subunit-3), Asp²⁶²-Phe¹¹³⁸ (subunit-1), and Val¹¹³⁹-Ser¹⁶⁸³ (subunit-2). HSTBP showed a weak similarity (29-40%) to clotting proteins of crustaceans and conserved vWF type D domain at Phe¹³⁸⁷-Gly¹⁵⁴⁴ in the subunit-2. The recombinant protein of the subunit-2 bound TTX at a molecular ratio of 1:1. Therefore, HSTBP may neutralize TTX and prevent the lethal toxicity of TTX. It could be applicable to possible antidotes for TTX intoxication.

Author Response File: Author Response.pdf