From the Light Chain Sequence to the Tissue Microenvironment: Contribution of the Mesangial Cells to Glomerular Amyloidosis
and Guillermo A. Herrera 1,*Round 1
Reviewer 1 Report
The manuscript entitled "From The Light Chain Sequence To The Tissue Microenvironment: Contribution Of The Mesangial Cells To Glomerular Amyloidosis" reviewed currently knowledge about why and how certain light chains are prone to forming amyloid. It is a very interesting issue which analyzed the structural and environment factors that drive the amyloid aggregation of the immunoglobulin light chains. Also, the role of mesangial cells in the amyloid deposition of light chains in the glomerular space is discussed. The paper was addressed in a very good manner. It is characterized by sections properly organized. The authors used references updated (~30%) and included the most recently works and experimental approaches such as ssNMR and cryo-EM. However, some concerns that are listed below.
Major concerns:
-The introduction section is good and summarized but the authors generalize some aspects to most of proteins. For example, …“Proteins acquire their functional capabilities through the folding, a complex process that confers the proteins a defined, generally compact, and stable tridimensional conformation known as the "native state”… which not include to intrinsically disordered proteins (IDPs). Several IDPs are involved in misfolding disease too. The same occurs with ...”protein aggregates are very toxic for cells, damaging them by different mechanisms”..., but what about of stress granules or condensate resulting from liquid-liquid phase separation, or other biological examples?, which are functional aggregate of proteins and nucleic acids. I suggest to introduce that this paper is address to globular human proteins, particularly to those associated to misfolding diseases.
-Considering the nature of this paper, some aspects of the AL amyloidosis such as physiological features, treatments or prevalence could be shortly discussed in the introduction.
-Tacking in account the name of Seccion 2. “Structural factors and environmental conditions”… I expected more details respect of environmental conditions that drive the amyloid aggregation of the LCs, more than lines 363-363. Renaming section 5, would allow to explore and to develop the environmental conditions topic. Also, in my opinion this name “What do we know about the mechanism of LC amyloid aggregation?” has been analyzed during all the text, and not only in section 5.
-What are the challenges involved in understanding and treating conformational diseases?
-At the end of some sections, the authors indicate what is unknown or what kind of experiments are required to answer some aspect of the AL amyloidosis. However, in your opinion, where should future research be directed in the near future to improve our understanding of AL amyloidosis? These perspectives, could be shortly discussed before the conclusions.
-In the conclusion it is worth including other factors discussed by you, more than ...” appears to depend on other factors, such post-translational modifications of the precursor, and proteolysis of the aggregate”, such as dynamic of proteins, micro-environment, etc.
-Edit all figures using the same styles for letters into them (size, type, color) and check the description of figures to homogenate the style of A), B). Use 1A, 1B, etc to do a reference of figures in the main text.
-Figures 5 and 6 can be merged, 6A and 6B as panel E and F, for example. Fig 6 only show more details of Fig 5.
- Figures 7, 8 and 9 can be merged. Figs 7 and 8 showed experimental demonstration of some steps described on Fig 8.
Minor concerns:
Eliminate some spaces between words from lines 61, 83, 161, 193, 255, 260, 273, 289, 318, 326, 419, 422, 472, 511, 524, 526, 528, 555, 815, 840.
Lines 68 and 79: Revise “amyloidoses”
Line 81: Check LCs in “immunoglobulin light chains (LC)”
Line 87: Reference as Figure 1A
Line 94 Reference as Figure 1A and 1B
Line 103: Eliminate point before “Structural factors….”
Line 120: Check “an LC”, LCs
Line130: Add “In other words,” the amyloid fibrils represents…
Line 161: Revise et al., cursive style.
Line 179-182: Add a verb to the sentence.
Line 183-185: Rephrase
Line 242: Separate it is not.
Line 246: LCs
Line 275 Use rVL
Line 292: Correct “in vivo” with italic.
Lines 304-305: Rephrase
Line 324: Remove capital letter “Like”
Line 327: Change the verb.
Line 362-364: Add references
Line 423: Correct “recombinant VL proteins (r)” with (rVL)
Line 479: Remove capital letter “In”
Line 554: Check sentence
Lines 578-584 Eliminate redundance
Line 582: Check “can al be”
Line 751: Check above world.
Line 810: Specify what is AA.
Line 917: Check figure1 description: Panel A and B are inverted; panel B in the figure refers Vλ, but appears as k LCs below. Also, homogenate VL (VL).
Line 937: Figure 2: Use black color for words and arrows.
Line 940: Add to description, the sequence of the green segment.
Line 942: Complete the description of the ex-vivo AL fibrils experiment showed.
Line 950: β-strand A is not showed in figure. To clear the figure the name of strands could be indicated with a letter on each one.
Line 955: Indicate CDR2 in panel A.
Line 965: “Three fibrillogenic fragments (Fibr fragments)”, needs to be represented clearer, for example in empty rectangles with the same color used in Figure 2B.
Line 972: Add reference. Check Yellow ovals.
Line 1001: were prepared
Line 1028: A clearer picture of Fig8A would be obtained by eliminating blue frame.
Line 1030: Check …“processing.in mesangial”… Add (MCs)
Line 1035 are filtered
Author Response
Reviewer 1
The manuscript entitled "From The Light Chain Sequence To The Tissue Microenvironment: Contribution Of The Mesangial Cells To Glomerular Amyloidosis" reviewed currently knowledge about why and how certain light chains are prone to forming amyloid. It is a very interesting issue which analyzed the structural and environment factors that drive the amyloid aggregation of the immunoglobulin light chains. Also, the role of mesangial cells in the amyloid deposition of light chains in the glomerular space is discussed. The paper was addressed in a very good manner. It is characterized by sections properly organized. The authors used references updated (~30%) and included the most recently works and experimental approaches such as ssNMR and cryo-EM.
Response to the reviewer
The authors thank the reviewer for the positive opinion about the content and structure of the manuscript.
However, some concerns that are listed below.
Major concerns:
-The introduction section is good and summarized but the authors generalize some aspects to most of proteins. For example, …“Proteins acquire their functional capabilities through the folding, a complex process that confers the proteins a defined, generally compact, and stable tridimensional conformation known as the "native state”… which not include to intrinsically disordered proteins (IDPs). Several IDPs are involved in misfolding disease too.
Response to the reviewer.
The authors agree with the reviewer's comment that the definition of protein folding used in the manuscript does not apply to the group of intrinsically disordered proteins. Considering the relevance of this class of proteins for cell physiology and that they are involved in diseases associated with protein aggregation, we have modified the introduction by adding texts (lines 36-45) that refer to the folding of intrinsically disordered proteins.
The same occurs with ...”protein aggregates are very toxic for cells, damaging them by different mechanisms”..., but what about of stress granules or condensate resulting from liquid-liquid phase separation, or other biological examples?, which are functional aggregate of proteins and nucleic acids. I suggest to introduce that this paper is address to globular human proteins, particularly to those associated to misfolding diseases.
Response to the reviewer.
The authors concur with the reviewer's comment that protein-nucleic acid complexes that give rise to stress granules or condensates resulting from liquid-liquid phase separation should be mentioned in this review. We have added texts to the manuscript. from lines 51 to 60, which address the aforementioned topic with some detail.
-Considering the nature of this paper, some aspects of the AL amyloidosis such as physiological features, treatments or prevalence could be shortly discussed in the introduction.
Response to the reviewer
The pathophysiology and treatment of AL amyloidosis are topics beyond the scope of this review. However, we agree with the reviewer that the manuscript does not address the incidence and prevalence of AL amyloidosis well. We have added text (lines 87-94) that refer to the epidemiology of the disease and its relation to misfolding and aggregation of light chains.
-Tacking in account the name of Seccion 2. “Structural factors and environmental conditions”… I expected more details respect of environmental conditions that drive the amyloid aggregation of the LCs, more than lines 363-363. Renaming section 5, would allow to explore and to develop the environmental conditions topic. Also, in my opinion this name “What do we know about the mechanism of LC amyloid aggregation?” has been analyzed during all the text, and not only in section 5.
Response to the reviewer
The authors agree with the reviewer's comment that the topic “environmental conditions promoting light chain amyloid aggregation" is not reviewed with the required depth in the manuscript. To address the reviewer's concern, a new section titled "3.6. Environmental factors modulating the propensity of LCs to form amyloid" was added from lines 422 to 457.
-What are the challenges involved in understanding and treating conformational diseases?
-At the end of some sections, the authors indicate what is unknown or what kind of experiments are required to answer some aspect of the AL amyloidosis. However, in your opinion, where should future research be directed in the near future to improve our understanding of AL amyloidosis? These perspectives, could be shortly discussed before the conclusions.
-In the conclusion it is worth including other factors discussed by you, more than ...” appears to depend on other factors, such post-translational modifications of the precursor, and proteolysis of the aggregate”, such as dynamic of proteins, micro-environment, etc.
Response to the reviewer
The general topic "conformational diseases" is beyond the scope of this review. However, the authors agree that the addition of a section addressing the most important challenges in AL amyloidosis research would add positively to the quality of the manuscript. To address the reviewer's concern, we added a new section titled “7. Challenges in AL amyloidosis research”. Additionally, some changes were made to the “Conclusions” section to address reviewer concerns.
-Edit all figures using the same styles for letters into them (size, type, color) and check the description of figures to homogenate the style of A), B). Use 1A, 1B, etc to do a reference of figures in the main text.
-Figures 5 and 6 can be merged, 6A and 6B as panel E and F, for example. Fig 6 only show more details of Fig 5.
Response to the reviewer
Figures 5 and 6 were merged and the new figure was numbered as figure 5. This modification was introduced in the main text.
- Figures 7 and 8 and 9 can be merged. Figs 7 and 8 showed experimental demonstration of some steps described on Fig 8.
Response to the reviewer
Figures 7 and 8 were merged and the new figure was numbered Figure 6. Figure 9 was kept as a separate figure to prevent Figure 6 from being too large and complex. Figure 9 has been renumbered as Figure 7. These changes have been made in the main text.
Minor concerns:
Eliminate some spaces between words from lines 61, 83, 161, 193, 255, 260, 273, 289, 318, 326, 419, 422, 472, 511, 524, 526, 528, 555, 815, 840.
Response to the reviewer
The extra spaces in the mentioned lines have been removed.
Lines 68 and 79: Revise “amyloidoses”
Response to the reviewer
“Amyloidoses” is plural of “amyloidosis”.
Line 81: Check LCs in “immunoglobulin light chains (LC)”
Done
Line 87: Reference as Figure 1A
Done
Line 94 Reference as Figure 1A and 1B
Done
Line 103: Eliminate point before “Structural factors….”
Done
Line 120: Check “an LC”, LCs
Done
Line130: Add “In other words,” the amyloid fibrils represents…
Response to the reviewer
With the purpose of improving the wording of the text that start on line 130, it has been rephrased as follows: “According to this notion, a major force driving the assembly of peptides and proteins in amyloid fibrils would be the intrinsic propensity of the polypeptide backbone to assemble into periodic structures”.
Line 161: Revise et al., cursive style.
Done
Line 179-182: Add a verb to the sentence.
Response to the reviewer
Ruiz-Zamora et al. used two complementary strategies to increase the probability to identify amyloid-forming segments in 6aJL2 protein
Line 183-185: Rephrase
The paragraph was rephrased as follows: The second strategy was based on the proteolysis of the 6aJL2 protein with trypsin, seeking to generate peptide fragments capable of forming amyloid-type fibrils, indicative of the presence of pro-fibrillogenic sequences.
With the purpose of improving the wording of the text that begins on lines 192-195, it was rephrased as follows: “It was shown that the aggregation of this fragment depends on the preservation of the disulfide bond Cys23-Cys88, a feature that could be a reminiscence of the structural restrictions that operate in the aggregation of the intact VL domain.
Line 242: Separate itis not.
Done
Line 246: LCs
Done
Line 275 Use rVL
Response to reviewer
The word “recombinant” is first used in the main text on line 275, and it was at that place of the text that we introduced (r) as an abbreviation for the word.
Line 292: Correct “in vivo” with italic.
Done
Lines 304-305: Rephrase
Response to the reviewer
The paragraph in lines 304-305 was rephrased as follows: “The frequency of this gene segment was also found to differ significantly between non-IgM and IgM ALl amyloidosis cases. It caused approximately 18% of non-IgM ALl cases, but only 2% of those classified as IgM ALl”.
Line 324: Remove capital letter “Like”
The phrase "LC folds into two domains" is a typo and has been removed. The correct sentence begins with "Like other proteins..."
Line 327: Change the verb.
Response to the reviewer
With the purpose of improving the wording of the paragraph spanning lines 192 to 195, it was rephrased as follows: “To deal with this risk, LCs have incorporated several structural elements that protect the edge strands from edge-to-edge interactions, an evolution-driven strategy that has been called "negative design"
In order to put the sentence on lines 379-381 in better context and make the idea expressed in it more understandable, it was moved to lines 323-325 and rephrased as follows: " This finding is in agreement with in vitro studies that have shown that mutations targeting structural residues at the beta-strand A of the VL, as Pro residues located in the kink, are particularly effective in promoting LC amyloid aggregation”.
With the purpose of improving the wording of the paragraph spanning lines 382 to 384, it was rephrased as follows: “There is experimental evidence suggesting that VL structural elements with a protective role are involved in the conformational adjustments that trigger aggregation”.
Line 362-364: Add references
Response to the reviewer
References were added
Line 423: Correct “recombinant VL proteins (r)” with (rVL)
Done
Line 479: Remove capital letter “In”
Done
Line 554: Check sentence
Response to the reviewer
The paragraph in lines 554-557 was rephrased as follows: “ssNMR analysis of 15N:13C differentially labeled samples determined that the monomers assemble into fibrils by stacking one on top of the other, in such an ordering that the protein segments in beta-strand form parallel in-register intermolecular beta-sheets
Lines 578-584 Eliminate redundance
Response to reviewer
The paragraph spanning lines 578-584 was rephrased as follows: “Thus, the authors concluded that the saline bridge Arg49-Asp25, generated by a somatic mutation, stabilizes the structure of FOR005 amyloid-like fibrils”.
Line 582: Check “can al be”
Done
Line 751: Check above world.
Response to the reviewer
It was changed to “aforementioned”
Line 810: Specify what is AA.
Response to the reviewer
It was defined as “serum amyloid A protein-derived (AA) amyloidosis”.
Line 917: Check figure1 description: Panel A and B are inverted; panel B in the figure refers Vλ, but appears as k LCs below. Also, homogenate VL (VL).
Response to the reviewer
The figure 1 description was corrected according to the suggestion of the reviewer.
Line 937: Figure 2: Use black color for words and arrows.
Response to the reviewer
Figure 2 was corrected according to the suggestion of the reviewer
Line 942: Complete the description of the ex-vivo AL fibrils experiment showed.
Response to reviewer
The description of figure 2 was modified, adding texts that explain in more detail the images shown and the purpose pursued with them. In addition, the references of the cited information were included.
Line 950: β-strand A is not showed in figure. To clear the figure the name of strands could be indicated with a letter on each one.
Response to the reviewer
The description of figure 3 was modified to eliminate potential ambiguity regarding the position in the structure of the 6aJL2 protein of the structural elements with a protective function. The beta-strand A is indicated in the figure.
Line 955: Indicate CDR2 in panel A.
Response to the reviewer
The perspective of the 6aJL2 3D structure shown in Figure 3A places CDR2 at the back of the image, making it difficult to indicate the position of CDR2. For reference, the CDR2 is indicated in panels B and C.
Line 965: “Three fibrillogenic fragments (Fibr fragments)”, needs to be represented clearer, for example in empty rectangles with the same color used in Figure 2B.
Response to the reviewer
The description of figure 4 was modified to eliminate potential ambiguity regarding the identity of the fibrillogenic fragments of protein 6aJL2. The relevant references were cited.
Line 972: Add reference. Check Yellow ovals.
Response to the reviewer
Done. Reference included in the figure description
Line 1001: were prepared
Done
Line 1028: A clearer picture of Fig8A would be obtained by eliminating blue frame.
Response to reviewer
Panel A of figure 8 was deleted and panel B was merged with figure 7 and the new figure was numbered as figure 6.
Line 1030: Check …“processing.in mesangial”… Add (MCs)
Done
Line 1035 are filtered
Done
Reviewer 2 Report
The review by del Pozo-Yauner et al addresses the genetic and structural characteristics, environmental conditions that drive amyloid aggregation of the immunoglobulin light chains (including a summary of most recent publications) and in particular the role of mesangial cells in the amyloid deposition in the glomerular space. The review is well written, all mechanisms (especially the “amyloid stetch hypothesis”) are well explained and understandable with minor linguistic/format issues that need to be addressed:
· Sentences starting with “but” should be avoided (starting with page 1, line 38, also page 11, line 521, also page 13, line 674, also page 14, line 716)
· Page 6, line 267: Sentence starting with “What determine such a…” is unclear
· Page 7, line 324: Change “Like” to “like”
· Page 7, Line 340: Change Pro to pro
· Page 12, line 582: sentence starting with “This study suggest…” is unclear
References should all be according to journal style (title in bold?)
Overall, congratulations to this important and very thorough review.
Author Response
Reviewer 2
The review by del Pozo-Yauner et al addresses the genetic and structural characteristics, environmental conditions that drive amyloid aggregation of the immunoglobulin light chains (including a summary of most recent publications) and in particular the role of mesangial cells in the amyloid deposition in the glomerular space. The review is well written, all mechanisms (especially the “amyloid stretch hypothesis”) are well explained and understandable with minor linguistic/format issues that need to be addressed:
- Sentences starting with “but” should be avoided (starting with page 1, line 38, also page 11, line 521, also page 13, line 674, also page 14, line 716)
Response to the reviewer
Sentences beginning with "but" were corrected by using "however" instead.
- Page 6, line 267: Sentence starting with “What determine such a…” is unclear
Response to the reviewer
The sentence was rephrased as follows: “The determinants of such a skewed repertoire of VL gene segments in AL amyloidosis remain unknown”.
- Page 7, line 324: Change “Like” to “like”
Response to the reviewer
The phrase "LC folds into two domains" is a typo and has been removed. The correct sentence begins with "Like other proteins..."
- Page 7, Line 340: Change Pro to pro
Response to the reviewer
We have found that the three-letter symbols for amino acids are often written with only capital letters, or with only the first letter capitalized, or with only lowercase letters, both in scientific publications and on biotech company websites. However, IUPAC recommends writing them as one capital letter followed by two lower-case letters, e.g. Gln (not GLN or gln), regardless of its position in a sentence or structure (https://iupac.qmul.ac.uk/AminoAcid/A1416.html). We used the IUPAC-recommended symbols because it makes reader confusion less likely.
Page 12, line 582: sentence starting with “This study suggest…” is unclear
Response to the reviewer
The typo in this sentence was corrected.
- References should all be according to journal style (title in bold?)
Response to the reviewer
Reference was formatted according to the journal style. We used the bibliography style for MDPI ACS Journals available in EndNote.
- Overall, congratulations to this important and very thorough review.
The authors greatly appreciate the words of the reviewer.
Reviewer 3 Report
The authors summarized the characteristics of LCs in terms of native structure, fibril structure, and also genetics producing various types of LCs. Especially the differences between native folding and fibril structure are well-summarized and provide readers useful information. This review will help guide the understanding of the mechanisms of LC amyloid formation and the development of therapeutic and diagnostic agents targeting AL amyloidosis. The manuscript is very well-written and worthy of publication.
Specific comments
- The authors mentioned that mutations modify the aggregation propensity. Intrinsic aggregation propensity, which is predicted from amino acid sequences, is not affected so much by mutations, especially single amino acid substitutions. Indeed, the most important segment near CDR1 has high amyloid score in all mutants. In the case of LCs, amyloidogenic sequences are hidden in the native fold. So, it seems that the stability and dynamics of native fold is more important than its sequence. Do you have any comments about this point?
- Recent cryo-EM development revealed several polymorphisms of fibrils. The readers may want to know the relationship between fibrils structure and pathological features on AL amyloidosis. If there is a literature about this point, it might be helpful.
Minor comments and Typos
Figure quality is not that great. It is suggested to improve the figures.
Following sentence should be reconsidered. In line 179, “Ruiz-Zamora et al. two complementary strategies to increase the probability to identified amyloid-forming segments in 6aJL2 protein.” Ruiz-Zamora et al. used/employed/developed ~~~
In line 654, Ans -> Asn
Author Response
Reviewer 3
Comments and Suggestions for Authors
The authors summarized the characteristics of LCs in terms of native structure, fibril structure, and also genetics producing various types of LCs. Especially the differences between native folding and fibril structure are well-summarized and provide readers useful information. This review will help guide the understanding of the mechanisms of LC amyloid formation and the development of therapeutic and diagnostic agents targeting AL amyloidosis. The manuscript is very well-written and worthy of publication.
Specific comments
- The authors mentioned that mutations modify the aggregation propensity. Intrinsic aggregation propensity, which is predicted from amino acid sequences, is not affected so much by mutations, especially single amino acid substitutions. Indeed, the most important segment near CDR1 has high amyloid score in all mutants. In the case of LCs, amyloidogenic sequences are hidden in the native fold. So, it seems that the stability and dynamics of native fold is more important than its sequence. Do you have any comments about this point?
Response to the reviewer
I agree with the reviewer that the impact on the thermodynamics of the native folding is likely the most important mechanism by which the somatic mutations modulate the amyloidogenesis of LCs. I think this is determined, at least in part, by the characteristics of the amyloid state. The cryo-EM structure of ex-vivo AL fibrils revealed that the incorporation of the LC into the fibrillar structure requires that it undergo an extensive conformational change that transforms it from a globular conformation to a quasi-bidimensional structure. Such structural rearrangement, which is the critical step of the mechanism of LC amyloidogenesis, is driven by the thermodynamic stability of the native state. Thus, mutations that compromise the thermodynamic stability of the native state, or increase the flexibility of regions of the LC whose conformational rearrangement determines the early stages of aggregation, will have a deep effect on LC aggregation. Furthermore, the cryo-EM structures of AL fibrils also suggest that the stability of these aggregates depends primarily on the network of intermolecular H-bonds between the atomic groups of peptide backbone of adjacent monomers. Although the interactions between the amino acid side chains also contribute to the stability of the amyloid fibrils and even can influence the topology of the polypeptide chain in the fibrillar state, the network of intermolecular H-bonds appears to be the main stabilizing force. This confers plasticity and robustness to the amyloid fibrils. That is, the ability to accommodate mutations that unfavorably alter the sequence pattern of pro-fibrillogenic regions of the LC without a large impact on fibril stability, through adjustments of the topology of the polypeptide chain. This is the main difference between the native state and the amyloid state of the LC.
On the other hand, evidence indicates that the VL domain contains several amyloidogenic hot spots and that most of them participate in the intermolecular contacts that trigger amyloid aggregation. This could explain why a single amino acid substitution, even when it significantly decreases the intrinsic aggregation propensity for a hot spot of the VL, will not abolish the ability of the entire VL to form amyloid. However, the effect will depend on the number of inhibitory mutations, the amino acids targeted by mutations, and the intrinsic aggregation propensity of the germline sequence. Finally, somatic mutations can impact the structural and physicochemical properties of the LCs in many different ways. Therefore, it is often not an easy task to determine what is the driving force behind the effect of a specific mutation on the amyloid aggregation of the LC. The effect of somatic mutations is context-dependent, and the context also includes other mutations present in the LC. This determines that the unfavorable effect of a mutation on the intrinsic propensity of the LC to form amyloid can be offset by its effect on the thermodynamic stability of the native state or flexibility of a certain region of the molecule. Similarly, one destabilizing mutation can counteract the inhibitory effect of another mutation on LC amyloidogenesis.
- Recent cryo-EM development revealed several polymorphisms of fibrils. The readers may want to know the relationship between fibrils structure and pathological features on AL amyloidosis. If there is a literature about this point, it might be helpful.
Response to the reviewer
It is logical to assume that AL fibrils with different structures also differ in pathological properties. Although it is a topic of the greatest importance to understand the pathogenesis of AL amyloidosis, to our knowledge, no studies have been published that explain the potential relationship between the structural characteristics of AL fibrils and their pathogenic properties. This is because, as the reviewer rightly mentions, the first structures of ex-vivo AL fibril determined by cryo-EM were only recently published. However, we briefly address this topic in a section that was added to the manuscript at the suggestion of reviewer 1, entitled Challenges in AL amyloidosis research.
Minor comments and Typos
- Figure quality is not that great. It is suggested to improve the figures.
Response to the reviewer
The original figures are tiff images with a resolution of 600 dpi. We use JPG images in the submitted manuscript to prevent the file from being too large.
- Following sentence should be reconsidered. In line 179, “Ruiz-Zamora et al. two complementary strategies to increase the probability to identified amyloid-forming segments in 6aJL2 protein.” Ruiz-Zamora et al. used/employed/developed ~~~
Response to the reviewer
The sentences mentioned by the reviewer were corrected.
In line 654, Ans -> Asn
This typo was corrected
