The Molecular Docking and Inhibition Kinetics of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Soft-Shelled Turtle Yolk
, Christoper Caesar Yudho Sutopo 2, Wei-Ting Hung 3, Ping-Hsun Wu 4,5,* and Jue-Liang Hsu 6,7,8,*
Round 1
Reviewer 1 Report
1. Did the authors perform any pH-related treatment of ACE and inhibitory peptides (PROPKA, etc.)? The protonation state of amino acids in enzymes and peptides will highly depend on the pH therefore the binding position and energy will be influenced.
2. Provide more details regarding the preparation of the ligands (inhibitory peptides) before the docking procedure.
4. Did the authors perform firstly "blind" docking to find the best-affinity place for ligands, or the binding place on the receptor was fixed before docking?
Author Response
Please see the attachment. Thank you so much!
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript describes the assessment of ACE inhibitory activities and mechanisms of peptides from soft-shelled turtle yolk which are reported as DPP-IV by the authors. The authors assessed five peptides and indicated the WLQL as a potent ACE inhibitory peptide with an IC50 value of 16.87 mM. Moreover, the authors succeeded to reveal a plausible mechanism. These results might be contribute to development of ACE inhibitors based on natural peptides. Thus, this paper is recommended for publication after minor revision.
Mainor revision
1. What is the main question addressed by the research?
The authors describe “LPSW can only bind to the non-active sites” (page10, line 345). I understand it means “ACE don’t cleavage LPSW”. However, The ACE inhibitor activity of LPSW increased with pre-incubation by ACE, and the fragmentation (digestion) of LPSW by ACE was detected with LC-MS analysis. The author should make sure of the inconsistency in the discussion of the mechanism of LPSW.
2. Do you consider the topic original or relevant in the field? Does it address a specific gap in the field?
The authors indicated novel peptides with ACE inhibitory activity, in this point this manuscript has originality and contributes to the field.
3. What does it add to the subject area compared with other published material?
See the answer to the 2nd question.
4. What specific improvements should the authors consider regarding the methodology? What further controls should be considered?
- The author should compare their peptides with positive control in ACE inhibitor activity.
- In ACE inhibitory activity assay with pre-incubation, WLQL, VPGLAL, LVGLPL and LPSW indicated an increase in ACE inhibitory activity. Moreover, the author revealed the amino acid sequence of the fragmented peptides with ACE using LC-MS analysis. The author should detect which peptide fragments derived from each ACEI peptide mainly contributes to ACE inhibition. And also do a docking study with each fragment.
- The authors should describe the preparation of synthetic peptides. The manuscript missing the method of synthetic experiment and characterization data of peptide. MS data is required at least.
5. Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed?
See the answer to the first question.
6. Are the references appropriate?
Appropriate references are cited in this manuscript.
7. Please include any additional comments on the tables and figures.
The author should add error bars for figure 1.
Figure 2 is unclear. The resolution should be improved and the vertical and horizontal magnification ratio should be the same.
8. Could you confirm a sentence on page 5 lines 190 to 193, in particular a part of “unlike LPSW and LPLF…”.
Author Response
Please see the attachment. Thank you so much!
Author Response File: Author Response.docx
Reviewer 3 Report
This manuscript investigated the ACE inhibitory activity of several DPP IV-inhibitory peptides previously derived from turtle yolk. Through some interesting results, it awaits further work to be published on this journal. Please see below detailed comments for the authors’ reference.
Abstract
Title better use turtle egg yolk?
Introduction
Line 40-41: note that these BP threshold values has been re-defined in some countries like the USA
Line 61-65: what is the importance/meaning of mentioning sACE and tACE here?
Line 70: here should clearly explain what structure motif of a DPP IV inhibitory peptide may be necessary for possessing ACE inhibitory activity?
MM
Line 135: here should be specify that is testicular ACE
Results & Discussion
Line 164-172: authors not only compared the peptides in the current study with those reported in literature but with very low activity, some recent papers, such as chicken [Fan & Wu (2021). Food Chem, 345, 128867] and egg [Fan, et al. (2019). J Agric Food Chem, 67(25), 7147-7156], and others reported very potent tetra-, and penta-, and hexa-peptides, which align with the peptide length in this study.
Line 20: note that proline is not an aromatic AA
Line 256: 1O8A not 108A
Line 256-260: molecular docking need to be re-run for two reasons: 1) lisinopril needs to be first docked to ACE to validate the accuracy of this current model [Fan & Wu (2021). Food Chem, 345, 128867], and 2) The hydrolyzed peptides after ACE pre-incubation such as WL and QL (from WLQL) as well as LP and SW (from LPSW) are suggested to dock with ACE, since these yielded dipeptides (which eventually inhibit ACE) showed higher ACE inhibition than their parent peptides.
I would also suggest authors to delete the docking results of LPLF and VPGLAL, which showed very weak ACEI activity, instead as mentioned above use WLQL (plus WL and QL) and LPSW (plus LP and SW) would be the major interesting peptides. That said, Tables 3-5 also need to be modified accordingly, as LPLF and VPGLAL will be eventually hydrolyzed into smaller peptides by ACE (as shown in Table 1); they actually aren’t able to form stable complex with ACE, so no need to be docked.
In addition:
1) LP and LF, hydrolyzed from LPLF by ACE, had no ACEI activity, while LF has been reported to possess strong ACEI activity in previous reported data [Wu, et al (2006). J Agric Food Chem, 54(3), 732-738], so please confirm the change of LPLF after ACE incubation.
2) WL from the same above paper [Wu, et al (2006). J Agric Food Chem, 54(3), 732-738] and possibly LP (that paper reported IP) may help you explain why WLQL and LPSW after ACE pre-incubation increased ACEI activity.
3) A big problem is to measure the ACEI activity of hydrolyzed peptides of LPSW and WLQL by ACE, since the newly-generated peptides showed higher activity than their parent peptides WLQL and LPSW. However, with the above discussion by leverage the ACEI activity reported in literature, authors may not need to synthesize WL, QL, LP, LF to confirm their activities.
Figure 3 seemed to be not necessary as this result has already been summarized in Table 1?
Some clues for the authors to explain the shared DPPIV and ACE inhibition, as the N-terminal of a peptide usually binds to DPPIV while C-terminal binds to ACE. This may be helpful to explain why some peptides possess both activities while others only possess one activity, as both terminal structures need to be look into
Author Response
Please see the attachment. Thank you so much!
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The authors addressed most of the comments. Still suggest having more comparison with peptides from literature especially those with similar structural features (Line 181-194), which will make the manuscript a more sound study.
Author Response
Comment 1:
The authors addressed most of the comments. Still suggest having more comparison with peptides from literature especially those with similar structural features (Line 181-194), which will make the manuscript a more sound study.
Response 1
According to your suggestion, we have already revised this part (Page 5, line 182-226). We hope that it gets your point. Thank you so much!
Author Response File: Author Response.docx
