Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2
, Mengqi Wang 1,†, Nan Wang 1, Caifeng Yang 1, Wenfang Guo 1, Gangqiang Li 1, Sumei Huang 2, Di Wei 2 and Dehu Liu 1,*
Round 1
Reviewer 1 Report
Peng et al present an analysis of the effects of differential glycosylation on the thrombolytic activity of the vampire bat salivary plasminogen activator alpha 2. While the data presented does appear interesting and the authors assessment that modulation of N398 and N185 glycosylation sites may well contribute beneficial therapeutic properties in terms of activity and fibrin specificity, respectively, the overall quality of presentation of the manuscript is unfortunately very poor.
The written style is very difficult to follow in a way that makes sense to the reader and the manuscript is strewn with minor errors:
On page 2, the authors state "DSPAα2 has the same glycosylation sites as t-PA, but its N-glycans are hybrid rather than high mannose." No supporting data or appropriate references are cited.
Also page 2, the authors mention "nucleotide" homology, referring to sequence alignments in Figures 1 and S2. Only amino acid alignments appear to be presented here.
Similar minor errors persist across subsequent pages but are too numerous to list individually. There are also substantial spelling/grammar and formatting errors throughout, particularly in figure legends.
The authors refer to supplementary figures that are not supplied - an SI word document appears to contain a Word version of the primary manuscript, while a separate pdf contains only gels of Figure S5... Where are figures S1-S4?
While the work may have potential for readers that are prepared to work hard to see the data, I believe a very substantial revision is required before publication can be considered.
Author Response
Dear reviewer 1:
Thank you for reviewing our manuscript and for the constructive comments, which greatly helped us to improve the manuscript. We have heavily revised our experiments and manuscript. The manuscript was carefully revised, and point-by-point response was listed below. We hope that your comments have been addressed accurately. The revised manuscript was marked with the “Track Changes” function and the responses were presented in red text.
Peng et al present an analysis of the effects of differential glycosylation on the thrombolytic activity of the vampire bat salivary plasminogen activator alpha 2. While the data presented does appear interesting and the authors assessment that modulation of N398 and N185 glycosylation sites may well contribute beneficial therapeutic properties in terms of activity and fibrin specificity, respectively, the overall quality of presentation of the manuscript is unfortunately very poor.
The written style is very difficult to follow in a way that makes sense to the reader and the manuscript is strewn with minor errors:
On page 2, the authors state "DSPAα2 has the same glycosylation sites as t-PA, but its N-glycans are hybrid rather than high mannose." No supporting data or appropriate references are cited.
Response: There was an error in this sentence, which has now been corrected (line 67-71).
DSPAα2 and t-PA share highly homologous N-glycosylation sites. However, the N-glycan chain of DSPAα2 expressed by Pichia pastoris is the high mannose type, while the t-PA expressed by mammalian cells is the hybrid type.
Also page 2, the authors mention "nucleotide" homology, referring to sequence alignments in Figures 1 and S2. Only amino acid alignments appear to be presented here.
Response: We are sorry for the omission of Figure S2, which have now been attached up.
Similar minor errors persist across subsequent pages but are too numerous to list individually. There are also substantial spelling/grammar and formatting errors throughout, particularly in figure legends.
Response: Thank you for your review, and for your feedback. We have rephrased the manuscript to aid clarity and think that it reads much better. To prevent confusion for the reader, we have tried to remove many of unnecessary statements. we took all your comments into account and revised these minor errors.
The authors refer to supplementary figures that are not supplied - an SI word document appears to contain a Word version of the primary manuscript, while a separate pdf contains only gels of Figure S5... Where are figures S1-S4?
Response: We are sorry for the omission of supplementary files, which have now been attached up.
While the work may have potential for readers that are prepared to work hard to see the data, I believe a very substantial revision is required before publication can be considered.
Response: We would like to thank you for your positive comments. We took all your comments into account and tried to respond and adapt the manuscript accordingly. We also improved the language wherever possible by a native English speaker.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer 2:
Thank you for reviewing our manuscript and for the constructive comments, which greatly helped us to improve the manuscript. We have heavily revised our experiments. The manuscript was carefully revised and point-by-point response was listed below. Since we didn't mark the line numbers in advance, some line numbers were wrong in response to your comment. We hope that your comments have been addressed accurately. The revised manuscript was marked with the “Track Changes” function and the responses were presented in red text.
It is a very good idea to check the influence of glycosylation on the activity and usability of this putative important protein. Removing and introducing glycosylation sites is a reasonable way to do so. So, the concept and the idea of the paper are fine.
However, the execution and performance are not adequate.
The conclusions drawn from the data presented are not comprehensible. Some chapters are
extremely sloppy written, with many copy-paste errors (worst example: legend to Fig1). It appears
that the paragraphs were written by different authors. Furthermore, the English is not sufficient and
due to many mistakes (singular/plural, wrong prepositions, wrong word order, missing commas,
missing verbs, random use of upper and lower case) very difficult to follow.
Technical challenges:
Manuscript under “Download manuscript” has no line numbering and is NOT exactly the same as the file in “Download supplementary file(s)”.
There are no other supplementary files containing additional figures. Just one with the original
gels/blots.
Comments in detail:
- In many places supplemental Figures are mentioned, but I do not see them (line 96, line 101,
…...)
Response: We are very sorry for such a problem. We have now attached these missed supplementary figures and tables to the supplementary file.
- Line 101: Really 96% nucleotide homology or amino acid homology??
Response: We are sorry for not expressing this clearly! DSPAα2 and DSPAα1 share 95.1% nucleotide homology and 89.5% amino acid homology. They are highly similar in the structure (line 212-214).
- Line 153: The proteins were modified, NOT the glycans
Response: Yes, the reviewer’s comment is right. We think that the expression was incorrect. We edited the statement at line 270-273.
- Legend Fig1. Copy paste errors, I see red and green arrows, but no blue ones.
Response: We have re-annotated Figure 1 and re-wrote the Legend of Figure 1.
- Fig.4a QNGlyalpha2 does not have any N-glycosylation sites. Why is it PNGase F sensitive?
Response: We have re-performed the SDS-PAGE experiment to improve the figure quality (Figure 4a). We increased the loading concentration of each sample while ensuring the same concentration of each sample. The positive bands on the lanes QNGlyα2+PNGase and QNGlyα2-2+PNGase can develop normally now, but the brightness of the bands is still significantly lower than Lanes QNGlyα2 and QNGlyα2-2 which have the same loading concentration. As a highly specific glucokinase, PNGase theoretically does not cleave non-specific sites, but experiments show that PNGase may also have the possibility of non-specific cleavage.
- Fig.4 In general, the shifts of molecular weight after incubation with PNGase F are enormous.
Check for the type of N-glycan produced in the Pichia expression system. Maybe they are
very bulky and influence therefore the protein usability a lot.
Response: as far as we know, among the current protein expression systems, the N-glycosylation modification of the Pichia pastoris expression system is the closest to mammalian. The N-glycan chain in Pichia pastoris is high mannose type (GlcNAc2Man2). The number of Mannose is generally 8-14. While the type of N-glycosylation in mammals is more complex, often including high mannose type, complex type or heterozygous type. This is also an important factor for Pichia pastoris as a potential heterologous protein expression system. Moreover, previous studies have shown that different N-glycosylation types only slightly affect the activity of DSPA. The main purpose of this study is to demonstrate that the absence or increase of N-glycosylation may affect the activity of DSPA, so we believe that the type of N-glycan is not so important for our study.
However, thanks for your suggestion, we think that sequencing and studying N-glycans may be a new research direction.
- Line 225: Why did you expect an increase of activity? A proper glycosylation may be necessary for an ideal conformation, but why should an additional glycan increase the activity?
Response: DSPAα2 shares 48% amino acid sequence homology with uPA. The two plasminogen activators are highly similar in structure, and both exert fibrinolytic activity in the form of a complete single chain. The N-glycan chain of u-PA is highly correlated with its serine protease activity. Considering the effect of glycosylation on activity, we speculated that N-glycan chains in the homologous region may have similar functions. Moreover, the N368 site is highly close to the active pocket of DSPAα2 in the structure. The active pocket is the centre of the serine protein activity of DSPAα2. Proper N-glycan chain introduction can make the structure of the active pocket more stable. Therefore, we predicted that the introduction of glycosylation site mutations in the homologous region of DSPAα2 might lead to the enhancement of its serine protease activity.
- Revise layout of Fig 7: typeface, size.
Response: We have adjusted the font, size, layout, etc. in the figure to the same level.
- Line 284-289: The data in Tab2 do not support these conclusions. Only QNGlyalpha2-2 is
significantly lower than the others.
Response: We are sorry that there was a conversion error when converting the relevant data of QNGlyα2. We have carefully compared the original experimental data and the data in the manuscript and confirmed that they are indeed errors. We have corrected the relevant data (Table 2, Line 461-471).
Both QNGlyα2 and QNGlyα2-2 are N398Q mutant proteins, so their serine protease activity should be at the same level, which is much lower than that of the original protein (rDSPAα2, QNGlyα2-1, ANGlyα2+1).
- Line 320-321: Data do not support this statement.
Response: We apologize again for our carelessness. Based on the original erroneous data, this conclusion is incorrect. We have carefully compared the original experimental data and the data in the manuscript and confirmed that they are indeed errors. We have corrected the relevant data.
The conclusion “The N398 site is highly correlated with the plasminogen activation activity of DSPAα2. After mutating N398 to Q398, the two related mutants(QNGlyα2，QNGlyα2-2) showed a large reduction in activity.” Now is obtainable and credible from the corrected data (Line488-490).
- Line 335-336: Sentence is incomprehensible.
Response: We apologized for the incomprehensible sentence. This sentence should be rewritten as “By using modification to humanize N-glycans, we can obtain highly active recombinant rDSPAα2 that is more suitable for humans using the Pichia pastoris expression system” (Line 521-528).
- Line 342-343: The protein (glycosylation site) has been modified, not the sugar chain.
Response: The statement in Line 528-530 refers to the modification of the N-glycosylation modification system in Pichia pastoris by a certain method, so that the N-glycan on the recombinant protein can avoid causing the immune response of human. There have been encouraging recent advances involving the glycoengineering of Pichia pastoris that have resulted in the humanizing of
the protein glycosylation pathways in this cell. We apologized for the misinterpretation due to the unclear formulation and have now reorganized the language to make this statement more precise.
- Conclusion: The presented data do not support the argumentation.
Response: â‘ Pichia pastoris is the most commonly used expression system, and its N-glycosylation modification system is the closest to the human among many expression systems. Therefore, using Pichia pastoris as a glycosylation research model is representative.
â‘¡ As a heterologous protein expression system, Pichia pastoris has been applied in various fields such as medicine and food, and has provided a variety of heterologous proteins with commercial value. As a medicinal protein, the industrial expression system of DSPAα2 will definitely be tried in Pichia pastoris. As far as we know, the expression level of DSPA in yeast is superior to other expression systems. Therefore, it is representative to use Pichia pastoris as a research model.
â‘¢ As mentioned，the N398 site is highly correlated with the plasminogen activation activity of DSPAα2. After mutating N398 to Q398, the two related mutants(QNGlyα2，QNGlyα2-2) showed a large reduction in activity.
â‘£ The N-glycan chain at the N185 site is involved in the recognition of fibrin. DSPAα2 is highly selective for fibrin, and its activity can be increased about 11 times when fibrin is involved in the experiment. However, when the N-glycan chain at this site was removed, its activity increase fold (up to 2 folds) was greatly reduced compared with the wildtype.
Above all, we believe that this conclusion is not problematic.
However, we believe that the reviewer's opinion has some value, and we add certain qualifications to the conclusion. For details, please refer to “Conclusion”.
- Methods: PNGase-digest is missing
Response: we are very sorry for such an error. We have added this part in “Materials and methods” section (Line 150-152).
- Methods: Carbohydrate staining of the gel is missing
Response: Sorry for missing this part. We have added this part in “Materials and methods” section (Line 147-150).
- Methods: Explain the abbreviation [pNA]
Response：[pNA]: A chromophore which can be cleavaged from S2765 by specific enzymes, free pNA can be detected by spectrophotometer or microplate reader to determine the activity of the corresponding enzyme.
- A careful language check of the whole manuscript is required, there are many many many
mistakes!
Response: Thank you for your suggestion. English has been edited by a native English speaker.
Other comments:
If you give additional information, a citation or figure information, include it within the sentence and
not after. The dot has to be AFTER the citation or figure. Line 59 … in the treatment of embolism
patients [1-3].
Response: Thank you for your comments! We have corrected them all according to your comments.
Several different typefaces.
Response: We have unified the font and format of the whole text.
Summary:
The idea is very good and should be pursued further. The basic strategy and methods are fine. In
order to be able to make an actual statement about the influence of glycosylation on the activity and
functionality of the protein, it is essential to at least estimate the size of the N-glycans involved (for
example: analysis of the released glycans after PNGase F degradation by mass spectrometry). It could
be that Pichia is not the ideal expression system, as the glycans there can often be very large and
bulky. Be more careful with the conclusions based on the data presented. Much more care and
effort should be put into the presentation of the data.
Response: Thank you for your kind patience. We have revised the manuscript in accordance with the comments and marked all the amends on our revised manuscript. We hope that all your comments have been addressed accurately.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The revised manuscript by Peng et al appears substantially improved in clarity and is now much easier for the reader to follow. The central conclusions that DSPAα2 can be produced in an active form in Pichia pastoris with maintenance of the critical N398 glycosylation site provides an interesting potential for therapeutic development.
While substantial improvements in the manuscript have been made, I still noticed a number of minor errors:
Page 2, line 59, correct 'wildy' to "widely"
Page 4, line 192, correct 'wild N-glycosylation...' to "wild-type N-glycosylation..."
Author Response
Dear reviewer 1:
Once again, thank you for reviewing this paper. We have heavily revised our manuscript again and corrected these problems with English syntax or incorrect words. The point-by-point response was listed below. We hope that your comments have been addressed accurately. The revised manuscript was marked with the “Track Changes” function and the responses were presented in red text.
The revised manuscript by Peng et al appears substantially improved in clarity and is now much easier for the reader to follow. The central conclusions that DSPAα2 can be produced in an active form in Pichia pastoris with maintenance of the critical N398 glycosylation site provides an interesting potential for therapeutic development.
While substantial improvements in the manuscript have been made, I still noticed a number of minor errors:
Page 2, line 59, correct 'wildy' to "widely"
Response: We have corrected this mistake (Page 2, line 65-66).
Page 4, line 192, correct 'wild N-glycosylation...' to "wild-type N-glycosylation..."
Response: We have corrected this linguistic error (Page 5, line 222).
Response: We have carefully reviewed the full text again and necessary corrections have been made in the text. The detailed corrections are listed below.
Page 3, line 116, corrected 'The three-dimensional structure of DSPAα2' to "The three-dimensional(3D) structure of DSPAα2”.
Page 3, line 117, corrected 'add N-glycosylation sites' to " add N-glycan chains”.
Page 5, line 200-201, corrected 'as the Optimal reaction pH 5.0' to "as the means of the optimal reaction temperature”.
Page 6, line 260, corrected 'when mutating these sites' to "when these sites were mutated”.
Page 7, line 287-288, corrected the format.
Page 11, line 382, corrected 'amd' to "and".
Page 11, line 391, corrected 'its center of activity' to "its active center".
Page 14, line 433, corrected 'the fibrin specificity' to " the fibrin sensitivity".
Page 15, line 438, corrected the format.
Page 15, line 440, corrected the format.
Page 15, line 444, deleted 'N-linked'.
Page 15, line 448, corrected the format.
Page 15, line 449, corrected the format.
Page 16, line 479-480, corrected 'fibrin specific recognition ability' to " fibrin-sensitivity".
Page 16, line 504, corrected 'seletive' to " sensitive".
Page 16, line 508, corrected ' Trp152, Tyr162, Trp190, Tyr192 and Leu201' to " W152, Y162, W190, Y192 and L201".
Page 17, line 545, corrected ''fibrin specificity ' to "fibrin sensitivity".
Page 17, line 550, corrected the format.
We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.
Author Response File: Author Response.docx
Reviewer 2 Report
The paper improved dramatically. The missing figures and informations are given, the language and the typing mistakes are corrected. Thanks to reviewer 1 also the finals typos have been corrected. I am still not really impressed by the paper, but now it can be published.
Author Response
Response to Reviewer 2 Comments
Dear reviewer 2:
Thank you very much for your suggestions and comments during review. Your comments have greatly helped us improve the manuscript. We have heavily revised our manuscript again and corrected these problems with English syntax or incorrect words. The revised manuscript was marked with the “Track Changes” function and the responses were presented in red text.
The paper improved dramatically. The missing figures and informations are given, the language and the typing mistakes are corrected. Thanks to reviewer 1 also the finals typos have been corrected. I am still not really impressed by the paper, but now it can be published.
Response: Thank you for your pertinent suggestions and affirmation of our modification. We hope to demonstrate the important influence of N-glycosylation sites on the activity of DSPAα2 through this study. Although this paper still has certain limitations in design and experiments, these limitations do not affect this conclusion. This manuscript can provide certain research ideas for the pharmacological research of DSPAα2. Thanks again for your comments and suggestions which helps us improve the manuscript greatly.
Kind regards,
Huakang Peng
2022.08.26
Author Response File: Author Response.docx
