Recombinant Aflatoxin-Degrading F420H2-Dependent Reductase from Mycobacterium smegmatis Protects Mammalian Cells from Aflatoxin Toxicity
Round 1
Reviewer 1 Report
Work prepared at a high level. The introduction, however, is too long and contains information obvious about Aflatoxins regarding their toxicity. the reviewer suggests shortening this information or deleting it. It is also unfortunate that the word "eco-friendly" is a short-cut, please substitute another expression. Figures are of poor quality, especially subtitles.
Author Response
Response 1: Thank you for your suggestion. For the first comment, “too long
introduction”, we revised the introduction of manuscript with a shorter description
about aflatoxin toxicity. For the second comment, “poor quality figure”, we will
upload higher quality figures at this revision.
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript at hand describes the reduced cytotoxic effect of aflatoxin B1 on a hepatic cell line by coincubation with a previously published highly active F420H2 dependent reductase (MSMEG_5998, FDR) from Mycobacerterium smegmatis. To this end the authors have constructed a Trx-FDR fusion protein and used it in combination with an established regeneration system for the cofactor to treat aflatoxin exposed cells. They report restored viability and the non-induction of apoptosis-related gene products.
Overall this manuscript is sufficiently well written, the experiments seem to be carried out correctly and are presented according to publishing standards. Yet the overall relevance of this paper is limited. Its findings do not warrant publication.
The activity of MSMEG_5998 is sufficiently well documented. The increased performance of the Trx-fusion is not worthwhile the lenghts the authors spend to elaborate on it. If there is more to it than increased stability/expression then the authors need to provide evidence for that other than increased activity, which could relate to stability issues between the tested variants (that have not been tested).
MSMEG_5998 is known to be poorly soluble (Taylor et al 2010) and solutions have been found elsewhere that suggest for this and other similar genes N-terminal modification greatly increase functional protein titers, either by N-terminal truncation (Lapalikur et al 2012) or fusion to maltose binding protein (doi: 10.1002/pro.2645). I lack the citation but remember that simply moving the His-tag from the C to the N terminus should also lead to improvements. None of these are discussed or more importantly compared to in the manuscript, to make a case for the improvements the Trx-fusion could add. If the Trx fusion as the authors suggest does not improve solubility but the conformational activity of the FDR then this needs to be documented (and compared to the high soluble N-terminal truncation from Lapalikur 2012).
Any follow up work comparing wt and fusion adds only to the speculation: The second main result reported concerns the reduced toxicity of Aflatoxin to sensitive cell culture if coincubated with a complex mix of FDR, the exotic cofactor and a regeneration system. Again the Trx-fusion was compared to the wildtype gene. Differences in the abundance of apoptosis-related gene products after 48 h but not 24h of incubation were documented. The toxicity assays were only performed for the Trx-fusion, we do not know how the wildtype gene would have performed. Again, my hypothesis is that the wildtype FDR is simply not stable enough under these conditions and this should be tested.
Either FDR does the same job: Transforming AfB1 into a reduced metabolite. This metabolite is not sufficiently well documented, but the catabolism following this crucial step is. It would have been nice to see the authors isolate the FDR product and use this in the toxicity assays and/or report on the products stability in the cell model by tracing the metabolites. Which protein variant works better in a highly complex set has comparably little relevance as the experimental setup has little implications for actual real life applications.
Author Response
Point 1: The activity of MSMEG_5998 is sufficiently well documented. The
increased performance of the Trx-fusion is not worthwhile the lenghts the authors
spend to elaborate on it. If there is more to it than increased stability/expression then
the authors need to provide evidence for that other than increased activity, which
could relate to stability issues between the tested variants (that have not been tested).
Response 1: Thank you for your suggestion. It is true that the activity of
MSMEG_5998 has been well documented; however, the protective effect of this
enzyme on mammalian cells has not been reported before. In this study, we found
Trx-linked MSMEG_5998 had better enzyme activity and better protective effects
than native one. We didn’t do any over-explanation according to our results. However,
it is still unclear whether the enzyme activity of Trx-linked MSMEG_5998 is better
due to its protein stability. We will perform protein stability comparison for native and
Trx-link MSMEG_5998 in the future but not in current work.
Point 2: MSMEG_5998 is known to be poorly soluble (Taylor et al 2010) and
solutions have been found elsewhere that suggest for this and other similar genes
N-terminal modification greatly increase functional protein titers, either by N-terminal
truncation (Lapalikur et al 2012) or fusion to maltose binding protein (doi:
10.1002/pro.2645). I lack the citation but remember that simply moving the His-tag
from the C to the N terminus should also lead to improvements. None of these are
discussed or more importantly compared to in the manuscript, to make a case for the
improvements the Trx-fusion could add. If the Trx fusion as the authors suggest does
not improve solubility but the conformational activity of the FDR then this needs to
be documented (and compared to the high soluble N-terminal truncation from
Lapalikur 2012).
Response 2: Thank you for your suggestion. According reviewer’s opinion, we have
added a paragraph “FDRs are known to be poorly soluble … these possibilities will be
addressed in the future.”in the discussion section to compare the effects of the
terminal modification and fusion to maltose binding protein of FDR on its protein
function, solubility, and stability. It is obvious that the enhanced ability of Trx-linked
MSMEG_5998 in degrading aflatoxin and protection may result from structural and
functional improvements or better protein stability but not from protein solubility. The
molecular details of these possibilities will be addressed in the future and are beyond
the scope of the current work.
Point 3: Any follow up work comparing wt and fusion adds only to the speculation:
The second main result reported concerns the reduced toxicity of Aflatoxin to
sensitive cell culture if coincubated with a complex mix of FDR, the exotic cofactor
and a regeneration system. Again the Trx-fusion was compared to the wildtype gene.
Differences in the abundance of apoptosis-related gene products after 48 h but not 24h
of incubation were documented. The toxicity assays were only performed for the
Trx-fusion, we do not know how the wildtype gene would have performed. Again, my
hypothesis is that the wildtype FDR is simply not stable enough under these
conditions and this should be tested.
Response 3: Thank you for your suggestion. It was our experimental design and we
didn’t do any over-explanation according to our results. There was no doubt that we
didn’t test cytotoxicity effects of native FDR. However, according to our results (Fig.
3 and 4), it was suggested that the protective effects of Trx-linked FDR was p53
pathway-dependent. The less p53 pathway activation meant more cell survival. Indeed,
the native MSMEG_5998 presented only minor changes of p53 pathway maker
proteins while Trx-link MSMEG_5998 did huge changes of them, indicating a better
protective function with Trx-link one (according to Fig. 5). For the reviewer’s
hypothesis about protein stability, we will test this possibility in the future.
Point 4: Either FDR does the same job: Transforming AfB1 into a reduced metabolite.
This metabolite is not sufficiently well documented, but the catabolism following this
crucial step is. It would have been nice to see the authors isolate the FDR product and
use this in the toxicity assays and/or report on the products stability in the cell model
by tracing the metabolites. Which protein variant works better in a highly complex set
has comparably little relevance as the experimental setup has little implications for
actual real life applications.
Response 4: Thank you for your suggestion. FDR transform AFB1 into a reduced
metabolite through breaking one double bond in the structure of AFB1. However,
based on previous work (Lapalikar 2012, PlosOne), the metabolite easily undergoes
spontaneous-hydrolysis which are unstable in vitro. Therefore, it is difficult to trace
and isolate FDR products in the cell culture model, and beyond the scope of the
current work. For the comment on real life application, this study only presented in
vitro results for cellular toxicity evaluation rather than data for real life application
purpose. If we want to make Trx-linked enzyme apply to real life in the future, we
have to do more modification and simplify our experimental conditions as mentioned
by reviewer. In addition, we should test its protective effects in animal models to fit
real situation.
Author Response File: Author Response.pdf
Reviewer 3 Report
The paper refers about the design, production and in vitro assay of a recombinant aflatoxin- degrading F420H2 dependent reductase, which would protect from aflatoxin toxicity. The manuscript is well written and interesting. Some minor concerns are listed below
line 25 - produced by Aspergillus species
line 35 - Aflatoxin M is a toxic metabolite of aflatoxins, which is found in milk in animals administered with feed containing aflatoxins. Please add this statement
line 80 - Escherichia coli per extensor
line 232 - E. coli (abbreviated and in italic)
Author Response
The paper refers about the design, production and in vitro assay of a recombinant
aflatoxin- degrading F420H2 dependent reductase, which would protect from
aflatoxin toxicity. The manuscript is well written and interesting. Some minor
concerns are listed below
line 25 - produced by Aspergillus species
line 35 - Aflatoxin M is a toxic metabolite of aflatoxins, which is found in milk in
animals administered with feed containing aflatoxins. Please add this statement
line 80 - Escherichia coli per extensor
line 232 - E. coli (abbreviated and in italic)
Response 1: Thank you for your suggestion. We agreed with reviewer’s suggestion
and revised line 25, 35, 80, and 232 of manuscript as the reviewer’s opinion.
Author Response File: Author Response.pdf
Reviewer 4 Report
General remarks:
Authors describe results of their study of fusion Trx-MSMEG_5998 protein in relation to AFB1 and its toxic effects on Hep cells. The study is interesting, and its results may possibly provide the development of the corresponding antidote to AFB1 (in the case of successful in vivo studies).
Good level of English, but, in my opinion, sometimes the authors use too many “the”, and there are also some misprints, so probably a light language editing would be good.
Abstract
Line 13-14: probably it would be better to specify the difference in AFB1 degradation between Trx-linked and native proteins (give the numbers in the abstract, i.e., quantitative description of the effect).
Intro
Line 73-75: why did you choose thioredoxin to improve the solubility of FDR MSMEG_5998? The explanation should be present in this part of the manuscript. Why don’t move some phrases from the Results section to the Intro section (see the next comment)?
Results
Line 136-139: it seems it would be better to move this text into the Introduction section as the explanation of your choice of thioredoxin (see my previous remark).
Fig. 2B. I suggest it would be better to specify in the figure caption, which reaction is illustrated (native or recombinant protein).
Discussion
Line 234: “Furthmore…” I suggest it should be “Furthermore”.
Fig. S3. Figure caption: “The pH curve of native MSMEG_5998.” Please, clarify both the caption of this picture, and the Y-axis caption. Did you mean pH dependence of the AFB1-degrading activity?
Materials and methods
Line 395-396: “AFB1 levels were quantified by measuring the absorbance of ultraviolet light (365 nm) in a 96-well plate [41].” Did you mean HPLC? If not, then, please, specify the reference for such direct quantification at 365 nm. I do not see in this Ref. 41 any words about the direct measurement of plates under UV light. They quantified AFB1 by HPLC with detection at 365 nm. Your sentence looks like you just put a 96-well plate under UV detector; so readers may be confused. I suggest it would be better to mention in this subsection that residual AFB1 was quantified by HPLC (as the authors of the ref. 41 did). You describe HPLC conditions below in this section, so it would be enough just to mention HPLC here.
The same remark is for lines 131-132 (Fig. 2 caption) – you right about the direct measurement of absorbance at 365 nm – did you mean HPLC?
Author Response
Point 1: Good level of English, but, in my opinion, sometimes the authors use too
many “the”, and there are also some misprints, so probably a light language editing
would be good.
Response 1: Thank you for your suggestion. We agreed with reviewer’s suggestion
and revised manuscript with less “the”.
Point 2: Abstract
Line 13-14: probably it would be better to specify the difference in AFB1 degradation
between Trx-linked and native proteins (give the numbers in the abstract, i.e.,
quantitative description of the effect).
Response 2: We agreed with reviewer’s suggestion and revised abstract and the
related statement in result 2.2 of manuscript.
Point 3: Intro
Line 73-75: why did you choose thioredoxin to improve the solubility of FDR
MSMEG_5998? The explanation should be present in this part of the manuscript.
Why don’t move some phrases from the Results section to the Intro section (see the
next comment)?
Response 3: We agreed with reviewer’s suggestion and moved Line 136-139 from the
results section to the introduction section to explain the reason for the usage of
thioredoxin.
Point 4: Results
Line 136-139: it seems it would be better to move this text into the Introduction
section as the explanation of your choice of thioredoxin (see my previous remark).
Response 4: Please see previous response.
Point 5: Fig. 2B. I suggest it would be better to specify in the figure caption, which
reaction is illustrated (native or recombinant protein).
Response 5: We agreed with reviewer’s suggestion and “The same reaction of
Trx-linked MSMEG_5998” was mentioned in the legend of Fig. 2B.
Point 6: Discussion
Line 234: “Furthmore…” I suggest it should be “Furthermore”.
Response 6: We corrected this misprint at revised version of manuscript.
Point 7: Fig. S3. Figure caption: “The pH curve of native MSMEG_5998.” Please,
clarify both the caption of this picture, and the Y-axis caption. Did you mean pH
dependence of the AFB1-degrading activity?
Response 7: We agreed with reviewer’s suggestion and figure S3 caption had been
changed to “The pH dependence of the AFB1-degrading activity in native
MSMEG_5998”. The Y-axis caption was also corrected to “rate (μmole/min/μmole
native MSMEG_5998)”.
Point 8: Materials and methods
Line 395-396: “AFB1 levels were quantified by measuring the absorbance of
ultraviolet light (365 nm) in a 96-well plate [41].” Did you mean HPLC? If not, then,
please, specify the reference for such direct quantification at 365 nm. I do not see in
this Ref. 41 any words about the direct measurement of plates under UV light. They
quantified AFB1 by HPLC with detection at 365 nm. Your sentence looks like you
just put a 96-well plate under UV detector; so readers may be confused. I suggest it
would be better to mention in this subsection that residual AFB1 was quantified by
HPLC (as the authors of the ref. 41 did). You describe HPLC conditions below in this
section, so it would be enough just to mention HPLC here.
The same remark is for lines 131-132 (Fig. 2 caption) – you right about the direct
measurement of absorbance at 365 nm – did you mean HPLC?
Response 8: We really did the direct measurement of 96-well plate under 365 nm UV
detector. HPLC was not used in this part for simplicity. HPLC was only used in
experiment of Figure S3. The reason of citing this reference is to understand which
wavelength AFB1 will have the maximal absorbance at.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
none