Characterization of Lectin from Bauhinia holophylla Using Bioinformatics Tools

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled ‘Characterization of lectin from Bauhinia holophylla using bioinformatics tools’ evaluated the the physicochemical characteristics, structure, and functional properties of a Bauhinia holophylla lectin (BhL), sequenced from genomic material obtained from calli cultures, through bioinformatics tools. The in silico characterization of new legume lectins can provide valuable data about this large family of homologous proteins. However, I think the current manuscript still has a certain gap in terms of content and innovation for other researchers, and it is recommended to modify and resubmit it.

 

Major concerns:

(1) The author mentioned many times that in the Bauhinia genus, only less than 5% of the species' lectins have been isolated. Thus, what are the difficulties in extracting lectin from Bauhinia holophylla? The importance of studying the lectin in Bauhinia has not been explained?

 

(2) Most of the results are based on software predictions and lack the corresponding experimental data. In terms of manuscript capacity and innovation, it has not yet met the requirements for publication in Horticulturae.

 

(3) Considering the quality requirements for publishing papers in Horticulturae, I think the clarity of Figure 4, 5, 6, 9 should be improved.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer comments:

 

The manuscript entitled “Characterization of lectin from Bauhinia holophylla using bioinformatics tools”, has been reviewed and found Lectins are non-immunogenic proteins that play a pharmacological role and are involved in stress response. The objective of this study was to estimate the physicochemical attributes, structure, and functional attributes of a protein called Bauhinia holophylla lectin (BhL). Although the manuscript is well written and explains the in-silico analyses in detail. However, changes still need to be done to improve the quality of manuscript. Manuscript needs to be improved rigorously and based on my analysis I recommend major revision.

Major Revision:

1-     To establish the reliability of this study, it is necessary to conduct additional functional analysis, namely activity analyses. To obtain assistance for this objective you may refer to a relevant work, (DOI): https://doi.org/10.1016/j.adcanc.2023.100099.

Minor Revisions:

1-     What was used as a negative control for the reaction and why? Line # 162.

2-     Italicize the specie Caesalpinioideae subfamily. Line # 364.

3-     What is the confidence level, or score associated with the predicted post-translational modifications? Line # 390.

4-     Whether they are consistent with previous findings or represent novel observations? Line # 397.

5-     Experimental validation is crucial to confirm the presence and functional importance of these features. Line # 403.

6-     What kind of potential limitations of the computational predictions have you faced in transmembrane domains and structural characterization? Line # 727.

7-     The BLASTp results reveal a considerable discrepancy at the N- and C-terminal ends. You have stated that errors in previously deposited sequences are inevitable. How can you provide evidence to support the claim that your protein extraction and sequence identification methods are free from errors and hence trustworthy? Line # 500.

8-     In terms of structural stability, discuss the implications of a protein structure with fewer α-helices and more β-sheets. Do prospective benefits or drawbacks exist with regard to the folding, stability, or vulnerability of proteins to denaturation? Please elaborate in one to two sentences under the introduction section.

9-     BhL shows a low level of α-helix arrangements. Considering the biological effects of having a protein structure that is primarily composed of β-sheets. Does this structural configuration correspond to the protein's function or interactions within the cell?

1- Evaluate the potential influence of the prevalence of β-sheets on the function or activity of your protein. Do certain regions inside the β-sheets play a crucial role in attaching to other molecules or performing enzymatic reactions?

1- It would be beneficial for the authors to discuss any limitations of their study, such as potential biases in prediction or annotation, as well as any confounding factors that may have influenced the interpretation of the results. Addressing these limitations would enhance the transparency and credibility of the study.

Note: A comments file is also attached.

 

1

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Overall, the text is well-written in terms of the English language. Anyhow moderate editing of English language is needed.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

lines 54-83: This monolithic period is quite heterogeneous and should be broken into three sections:

1. Experimentally-related issues in understanding lectins
2. Increase in quality and quantity of bioinformatic tools/services
3. Use of these tools/services for advancing lectin knowledge with each section properly indented. Please pay attention especially to lines 75-77, which seem to be out of context in a paragraph related to general techniques.

line 42: The authors seem to imply that the Bauhinia genus is "one of the best investigated and characterized" in terms of lectins. At the same time, lines 84-86 state that "studies on Bauhinia lectins still remain scarce." I ask the authors to be clearer.

line 218: Please write explicitly that you use the default parameters (or clearly state the values you choose).

line 222: Write the correct URL for translate (https://www.expasy.org/resources/translate) and state the frame and the direction for the sequence chosen for blastp.

line 223: "comparison was verified" is not appropriate here: please explain better that you want to verify the sequence association with lectins. Besides, as in the case of blastn, write explicitly that you use the defaults (or state the parameter values you changed).

line 236: The authors use Clustal Omega to build the phylogenetic tree. Clustal Omega also produces an MSA. Why then do the authors use another tool (Multalin) to perform the MSA?

lines 261 and 265: Why do the authors use THMM and SignalP 4.1, given that the first is deprecated and the second is two versions before the current one? I suggest using https://dtu.biolib.com/DeepTMHMM. Besides, using InterProScan (which also refers to SignalP 5.0 and TMHMM 2.0) makes the procedure and the associated results section virtually useless.

lines 284-288: These lines are out of place in the results section.

line 344: Figure 2: Please explain what "!", "#", "%" stands for in the consensus sequence.

lines 353-354: The reduced length is because the sequence belongs to a PDB without the signal peptide.

line 354-357: Where does this information come from?

lines 357-359: This phrase is not useful in its current form.

line 359: I would write "The multiple sequence alignment" rather than "Analyses performed by the Multialin program."

line 363: It is "close," not "closer." Otherwise, the authors should include other lectins in the MSA and demonstrate that BhL is farther from these than from other Caesalpinioideae lectins.

line 366: The phylogram does not provide any useful information that cannot be inferred from blastp. So either add sequences or remove the phylogram.

line 375: (a) The scale has no units. (b) It is a pairwise alignment, not a multiple sequence alignment.

line 379: How does this data align with information from other lectins in Caesalpinioideae?

lines 391-395: The authors should note that YinOYang produces a warning related to the presence of a signal peptide.

line 399: Why is there no "threshold" line for YinOYang prediction despite the fact that the legend describes it?

lines 413-424: See the comment on lines 261 and 265.

lines 449-455: Are the authors aware that they are using an AlphaFold prediction with the signal peptide still ataced? Please either use alphafold2 via colabfold/deepming google colabs, or alphafold3 server, or 1gsl.1.A as template. In all cases authors need to add metrics supporting the result.

line 494: It is blastn, not blatn.

line 497: I cannot understand the reference to [45].

lines 501-503: See the comment on lines 353-354.

line 510: The word "catalytic" is misused here.

line 525: Clustal Omega is not an annotation tool. Furthermore, it gives a very preliminary indication of phylogenetic relationships. I suggest the authors limit their consideration to the MSA.

lines 531-534: I do not understand the logic behind this sentence: the importance/relevance of the conservation of catalytic/active/binding residues is not "suggested" by mutation. It is a fact, rather than a consequence. Mutation of these residues has effects that range from changes in specificity to changes in the reaction mechanism to the loss of catalytic activity.

lines 535-544: This statement is not supported by any results. Why is it here?

lines 545-560: This section should be rewritten to better compare the results associated with the in silico physicochemical characterization of BhL to other lectins.

lines 578-580: I would either like to see some structural analysis to support this affirmation (cavity size, surface charge distribution) or see the affirmation toned down.

lines 603-605: This sentence is not well integrated into the text, and removing it would not change the section in any way. Please either remove it or integrate it better.

line 611: Again, I do not think "evolutionary" is appropriate here.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made notable improvements to the manuscript in response to the reviewer's comments. This research is a valuable addition to the field of protein characterization and establishes the foundation for future research. It is anticipated that the subsequent studies will provide a comprehensive analysis of BhL, incorporating both computational and experimental approaches, as per the reviewers.

Overall, the manuscript is now of good quality and satisfies the requirements for publication in Horticulturae. I suggest acceptance of the manuscript with no further revisions required.

Comments on the Quality of English Language

Minor editing of English language is required e.g. Line # 13. This sentence in the abstract should be written in the present tense by the authors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I highly appreciate the effort the authors put in addressing my concerns.I find the new version more sound,understandable, and aligned with standard procedures. Nonetheless, I still have a concern related to the homology model.

My observation was that using an AF model as a template for homology modeling through SWISS-MODEL did not make sense. I suggested either ditching SWISS-MODEL in favor of AlphaFold or using a crystal structure (1gsl.1.A) as a modeling template. The authors rebutted by showing (very sound) AF3 results while saying that they prefer the SWISS-MODEL prediction based on the AF model. This does not actually address my concern. Please use the AF3 model OR generate a prediction using 1gsl.1.A as the template.

 

For the sake of cultural discussion, I also have two more question.

1. The author said: "MultiAlign has been shown to provide more accurate alignments in certain contexts, especially when dealing with complex or highly divergent sequences". I would like to have a reference for this.

2.  How do they map on the MSA/predict the proteinase cleavage sites? This information is nowhere to be found in the manuscript.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf