Crotoxin B from the South American Rattlesnake Crotalus vegrandis Blocks Voltage-Gated Calcium Channels Independent of Its Intrinsic Catalytic Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary:

 

The paper tried to present evidence showing that CB, a protein with phospholipase activity from the venom of C. vegrandis, inhibits Cav 2.2 and Cav 1.2 independent of its enzymatic activity.

 

Major Comments:

 

Lines 119 – 121: It is not clear whether the complete C. vegrandis CB is identified. Are the gaps in the alignment between C. vegrandis and C. durissus CB deletions or unidentified amino acids? If they are unidentified, why are they not identified? If they are not identified, I recommend getting the full sequence of C. vegrandis CB. I also recommend making the raw MS/MS data publicly available so that sequence identification can be independently verified.

 

Lines 221 – 224: Seems contradictory given that results from these experiments were concluded to have strong signal at the cell membrane. Line 223 mentions "capability of CB to be internalized" however this was not shown directly in these experiments. I think the evidence shown here is not enough to confidently conclude that CB is internalized. The method section did not mention any steps to quench fluorescence of labeled CB on the extracellular surface, thus results from these experiments are not enough to conclude that CB is internalized. I recommend doing these experiments and carefully quench the fluorescent label at the extracellular surface.

 

Lines 235 – 237: Please provide a rationale why only these two Cav's were tested. If you want to test pre-synaptic and post-synaptic channels why were Cav2.1 or Cav2.3 for presynaptic target and Cav1.3 for post-synaptic target tested?

 

Lines 249 – 250: What is the extent of cell damage at this concentration or at all the concentrations used for patch clamp recording? Figure 4A shows a high level of toxicity at 10 uM with barium. Could the inhibition of Cav be a result of the cytotoxic effect? What is the effect on another Cav? To show selective activity on Cav2.2 and Cav1.2, I recommend doing the same experiment on another Cav (e.g Cav 1.3, Cav2.1).

 

Lines 394 – 395: I think this conclusion is not supported by the data presented here. It is possible that the effects on Cav's are an indirect result of the cytotoxic effect of CB. Interestingly, the inhibitory effect correlates well with the cytotoxic effect. As the cytotoxic effect drops to 20%, the inhibitory activity against Cav also drops to 20% at 0.4 uM.

 

Lines 395 – 397: Internalization has not been shown here. What was shown was a possibility of internalization, but the experimental design did not eliminate another possibility that the signal from CB fluorescence could be exclusively coming from the protein bound to the cell surface. Please see comments for Lines 221 – 224.

 

 

 

Minor comments:

 

I think statements of the effects on the CNS are not well supported given that CB was not shown to cross the blood brain barrier (BBB) in vivo. If there are works showing that indeed this protein crosses the BBB in vivo, please cite those works in this paper.

 

Lines 43 – 45: Seems confusing given that line 37 mentions that CB is weakly toxic.

 

Lines 51 – 55: The effects of this protein were shown in an ex vivo synaptosome preparation thus, it is not clear whether this can cross the blood brain barrier and affect the CNS in vivo. Thus, saying that the protein affects the CNS is not well supported.

 

Line 82: Please be consistent with the name of the cell lines used. Here it says HEK 293 but, in the results section, tsa201 cells were used.

 

Lines 117 – 118: How specific is this antibody? Please provide a citation for the specificity of this antibody against CB. The dose dependent western blot is nice, but it would be better to run the same western blot with a negative control.

 

Lines 132 – 134: Can you say the same for C. vegrandis? Has this been shown previously? if so please provide a citation.

 

Lines 165 – 168: This statement is confusing. I get the impression that this analysis was done on C. vegrandis venom but lines 166-167 mentioned "corroborated the occurrence of CBc from C. durissus".

 

Line 262: Cav 2.2 or Cav 1.2?

 

Lines 310 – 311: Can this be confidently said about the activity in prey? I recommend that it should be stated here that this conclusion is derived from a heterologous expression system.

 

Lines 312 – 313: Is it also independent of other activity stated in lines 43-45.

 

Lines 359 – 360: Can CB cross the blood brain barrier (BBB)? I suggest discussion of the effect of this protein on the CNS should be excluded if this does not cross the BBB.

 

Lines 360 – 361: Has this been shown in vivo? If not, then rewrite this statement to reflect that this protein acts on channels with known post synaptic expression.

 

Lines 365 – 366: This can be due to the other effect of CB and not mainly due to the inhibition of Cav's.

 

Lines 367 – 369: For this to happen the protein must cross the BBB. Are there evidence indicating that CB can cross the BBB and if it does, is the behavior a result of Cav inhibition and not from the cytotoxic effect of venom?

Lines 384 – 386: Does this mean that the effect of CB on Cav's come from the products of the damage to the cell membrane and not directly from CB?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comments for author File: Comments.pdf

Author Response

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Reviewer 2 Report

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors I read the revised manuscript. Most of my major comments in the previous version were not addressed. These are:  

1. Controls to establish the internalization of C. vegrandis CB were not properly done hence it is not clear whether indeed it is internalized or remained bound to the extracellular side of the cell membrane.
2. The complete sequence was not identified. If trypsin digestion is not sufficient to provide the complete sequence, the authors can do other enzymatic digestion to fill the gaps in the sequence. This gives the impression that the current work is not thoroughly done. In addition, indeed there is some degree of confidence that what is presented here is CB because of its reactivity to the antibody from C. durissus however, the authors did not reference this work, and they failed to indicate that this antibody has cross reactivity across species. A complete sequence is important so that other researchers who may not have access to C. vegrandis venom can heterologously express this protein and verify the work presented here or perform further studies on this protein. 
3. The authors did not show that the channel inhibitory activity is independent of the cytotoxic activity of CB. It is important  to show this to support the claims presented in this paper.

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Reviewer 2 Report

Comments and Suggestions for Authors

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Comments on the Quality of English Language

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Author Response

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