Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and Sensitizes Neuroblastoma Cells to EGF Stimulated Proliferation
The abstract is well structured, but there are some points that could be improved to increase the understanding and fluency of the text.
Introduction:
Your introduction provides a broad overview of the context and motivation behind your study. The purpose of the research is quite clear. It is clearly explained that the study aims to investigate the role of oligomannose-type N-glycans in promoting the development and progression of neuroblastoma (NB). The problem of pediatric neuroblastoma is presented, with emphasis on the difficulties related to early diagnosis, drug resistance, and tumor recurrence. However, I recommend these suggestions:
1. "N-Glycosylation of proteins can modify numerous proteins, e.g., receptors, adhesion proteins, transporters, and ion channels, as well as extracellular proteins, which in turn regulate cell signaling events to control cellular properties, e.g., cell growth and invasion." This sentence lists many functions and proteins without clear links to how these modifications specifically affect NB. Explain in more detail and with concrete examples how N-glycan modifications specifically affect NB cell growth and invasion.
2. "A therapeutic target in many cancers is EGFR, a receptor protein implicated in cell proliferation and invasiveness. EGFR has been observed in NB tissues and is highly expressed in human NB cell lines."
The transition to the discussion of EGFR is a bit abrupt and could benefit from a more gradual transition that better connects the topic of N-glycans with that of EGFR. Introduce EGFR in the context of the N-glycan discussion, explaining how EGFR glycosylation is relevant to NB.
The points to be further clarified are:
3. Details on specific issues related to the diagnosis and treatment of NB.
4. Concrete examples of how glycosylation modifies protein functions relevant to NB.
5. Clearer transition between the N-glycan discussion and the introduction of EGFR.
Discussion:
The discussion presents some areas that could be further clarified to improve understanding and interpretation of the results. The specific points that need clarification and why they are important are:
1. It is not clearly explained why the reduction in MGAT1 expression was chosen and how this relates to previous studies in rats. Provide a more detailed explanation of the choice of MGAT1 and how this manipulation relates to previous studies, explaining the importance of this protein in N-glycosylation and the progression of NB.
2. Provide a detailed description of the mechanism by which frameshift mutations affect GnT-I activity and the balance between oligomannose and complex N-glycans. Explain how the replacement of complex N-glycans with oligomannose on EGFRs affects their function and cellular localization, linking these effects to the observed changes in NB cell proliferation and invasion.
3. The explanation of the effect of EGF on cell proliferation is a bit confusing and requires further detail. Elaborate on how EGF stimulation specifically influences cell proliferation in BE(2)-C(-MGAT1) compared to BE(2)-C, and why this is significant.
4. The explanation of why 3D cultures are more relevant than 2D cultures could be further explored. Highlight how 3D cultures better mimic the tumor microenvironment and why this is important for studying NB.
5. Describe in more detail the contradictions present in the studies on the role of RhoA in NB and provide an interpretation of the results of the present study in the context of these contradictions.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Comments to Reviewer 1
Thanks for your comments as the paper has been strengthened with regards to the introduction and discussion.
Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and Sensitizes Neuroblastoma Cells to EGF Stimulated Proliferation
The abstract is well structured, but there are some points that could be improved to increase the understanding and fluency of the text.
With the word limit, it is difficult to enhance the understanding to a general audience.
Introduction:
Your introduction provides a broad overview of the context and motivation behind your study. The purpose of the research is quite clear. It is clearly explained that the study aims to investigate the role of oligomannose-type N-glycans in promoting the development and progression of neuroblastoma (NB). The problem of pediatric neuroblastoma is presented, with emphasis on the difficulties related to early diagnosis, drug resistance, and tumor recurrence. However, I recommend these suggestions:
- "N-Glycosylation of proteins can modify numerous proteins, e.g., receptors, adhesion proteins, transporters, and ion channels, as well as extracellular proteins, which in turn regulate cell signaling events to control cellular properties, e.g., cell growth and invasion." This sentence lists many functions and proteins without clear links to how these modifications specifically affect NB. Explain in more detail and with concrete examples how N-glycan modifications specifically affect NB cell growth and invasion.
See paragraph 2 of the introduction.
- "A therapeutic target in many cancers is EGFR, a receptor protein implicated in cell proliferation and invasiveness. EGFR has been observed in NB tissues and is highly expressed in human NB cell lines."
This has been clarified as the sentence has been revised.
The transition to the discussion of EGFR is a bit abrupt and could benefit from a more gradual transition that better connects the topic of N-glycans with that of EGFR. Introduce EGFR in the context of the N-glycan discussion, explaining how EGFR glycosylation is relevant to NB.
A sentence was added to EGFR introduction paragraph to ease the transition into the discussion of EGFR.
The points to be further clarified are:
- Details on specific issues related to the diagnosis and treatment of NB.
Rephrased the first paragraph to focus on that NB tumors are commonly not diagnosed until metastasis has already occurred. Also changed the sentence structure to focus on half of high-risk patients will face life altering side effects which is why we need improved therapies.
- Concrete examples of how glycosylation modifies protein functions relevant to NB.
This has been added, see point 1, above.
- Clearer transition between the N-glycan discussion and the introduction of EGFR.
This has been addressed.
Discussion:
The discussion presents some areas that could be further clarified to improve understanding and interpretation of the results. The specific points that need clarification and why they are important are:
- It is not clearly explained why the reduction in MGAT1 expression was chosen and how this relates to previous studies in rats. Provide a more detailed explanation of the choice of MGAT1 and how this manipulation relates to previous studies, explaining the importance of this protein in N-glycosylation and the progression of NB.
This has been stated in paragraph 3 of the introduction and last paragraph of introduction with edits. Further all raised points clarified in first paragraph of discussion.
- Provide a detailed description of the mechanism by which frameshift mutations affect GnT-I activity and the balance between oligomannose and complex N-glycans. Explain how the replacement of complex N-glycans with oligomannose on EGFRs affects their function and cellular localization, linking these effects to the observed changes in NB cell proliferation and invasion.
These issues have been addressed in 1st paragraph and a mechanism proposed in 3rd paragraph of discussion.
- The explanation of the effect of EGF on cell proliferation is a bit confusing and requires further detail. Elaborate on how EGF stimulation specifically influences cell proliferation in BE(2)-C(-MGAT1) compared to BE(2)-C, and why this is significant.
This issue has been addressed, see 2nd paragraph in discussion.
- The explanation of why 3D cultures are more relevant than 2D cultures could be further explored. Highlight how 3D cultures better mimic the tumor microenvironment and why this is important for studying NB.
This point is addressed in 2nd and 3rd paragraphs, and more directly and explicitly in 4th paragraph of discussion.
- Describe in more detail the contradictions present in the studies on the role of RhoA in NB and provide an interpretation of the results of the present study in the context of these contradictions.
This has been addressed, see last paragraph in discussion. There was not a direct measurement of RhoA which could be done. Further refs were provided showing that ROCK proteins have opposing roles. Another point is the use of 2D versus 3D cell culturing conditions.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
By scoring Novelty and Significance of content as "average" this is not to be construed as a negative. Read "average" as good content worth publishing and reading, but not astounding or paradign shifting.
The subject of N-N-glycosylation is important and currently underappreciated in oncology.
After a necessary rewrite, this paper will be a small but welcome step in the right direction to further awareness of N--glycosylation’s importance in NB and in cancer generally.
Although medical English use is generally good, grammatically and idiomatically correct, this paper requires a comprehensive rewrite. It has not been proofread. There are numerous obviously omitted words, strange sentences, and vagueness. These errors are so numerous that I stopped enumerating them after five such errors in the first 50 lines.
Line 2, title might be better without the word “sensitizes”. Although using “sensitized” is correct as it stands, in current usage sensitized usually refers to increasing effect of a drug. Would “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF Stimulated Proliferation” work better ? Also the title must specify in vitro. So, “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma in vitro Invasiveness and EGF Stimulated Proliferation”.
Line 34. dont be vague, “which is unsatisfactory due to” . Tell us approximately what the median 5 yr OS or some OS range. A 5 year OS of 99% is unsatisfactory too.
Line 36, “of solid NB” ? What other kind of NB is there ? NB is not a leukemia.
Line 38, You are not going to “To improve the quality of NB patients…” They are beautiful, children, equal to any other children.
Line 40 should start a new paragraph.
Line 49 errors. You are addressing a wide variety of readers, some are clinician-researchers. They will most likely not remember exactly N-glycosylation steps. A figure here is required. Also note that the correct use is N-glycosylation, with lower case g. N-glycans, oligomannose, hybrid, and complex all must be defined in words and shown in a figure. Most of your readers will require reminders here.
I stopped enumerating errors at line 50. They do, however, continue throughout the manuscript.
Line 62, it might be ok to use EGFR without first spelling it out, it might be preferable though to mention its synonyms HER1 and ErbB1. Any discussion of EGFR must have a paragraph or two giving the broad outline of this receptor, its proclivity for heterodimerization, the oddity of EGFR overexpressing EGFR cancers not being slowed down by potent and effective EGFR inhibition, of universal development of resistance to EGFR inhibitors in those cancers initially responsive to EGFR inhibiting drugs, etc.
Line 72. As in comment above re. Line 32, vagueness, “Further the N-glycans of EGFR have been shown to contribute to both the function and regulation of EGFR…”.what does that mean ? you should tell us what - how effect function, what regulation, inhibit, stimulate or what ?
Do the authors think that a Table showing definitions and proliferative effects of BE(2)-C,
BE(2)-C +GnT-I, BE(2)-C(-MGAT1), and BE(2)-C(+/-MGAT1) would be useful ? Glycobiology is important yet somewhat neglected currently. Most readers might benefit from better didactic in your paper.
Line 266, does less processed mean smaller mol. wt. glycans, or fewer in number, or less branched, or what ?
Line 346, please forgive if I am wrong here, but I dont think autophosphorylation is essential for normal or pathological EGFR function. As heterodimer partner to a wide variety of non HER partners - over a dozen are currently recognized - EGFR can become phosphorylated and signaling active when the heterodimer partner has been liganded.
Options consideration: Do the authors think that work on N-glycans in cancer already from 1974 is worth mentioning ? [Oncology. 1974;29:249–264. doi:10.1159/000224907.]
By scoring Novelty and Significance of content as "average" this is not to be construed as a negative. Read "average" as good content worth publishing and reading, but not astounding or paradign shifting.
The subject of N-N-glycosylation is important and currently underappreciated in oncology.
After a necessary rewrite, this paper will be a small but welcome step in the right direction to further awareness of N--glycosylation’s importance in NB and in cancer generally.
Although medical English use is generally good, grammatically and idiomatically correct, this paper requires a comprehensive rewrite. It has not been proofread. There are numerous obviously omitted words, strange sentences, and vagueness. These errors are so numerous that I stopped enumerating them after five such errors in the first 50 lines.
Line 2, title might be better without the word “sensitizes”. Although using “sensitized” is correct as it stands, in current usage sensitized usually refers to increasing effect of a drug. Would “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF Stimulated Proliferation” work better ? Also the title must specify in vitro. So, “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma in vitro Invasiveness and EGF Stimulated Proliferation”.
Line 34. dont be vague, “which is unsatisfactory due to” . Tell us approximately what the median 5 yr OS or some OS range. A 5 year OS of 99% is unsatisfactory too.
Line 36, “of solid NB” ? What other kind of NB is there ? NB is not a leukemia.
Line 38, You are not going to “To improve the quality of NB patients…” They are beautiful, children, equal to any other children.
Line 40 should start a new paragraph.
Line 49 errors. You are addressing a wide variety of readers, some are clinician-researchers. They will most likely not remember exactly N-glycosylation steps. A figure here is required. Also note that the correct use is N-glycosylation, with lower case g. N-glycans, oligomannose, hybrid, and complex all must be defined in words and shown in a figure. Most of your readers will require reminders here.
I stopped enumerating errors at line 50. They do, however, continue throughout the manuscript.
Line 62, it might be ok to use EGFR without first spelling it out, it might be preferable though to mention its synonyms HER1 and ErbB1. Any discussion of EGFR must have a paragraph or two giving the broad outline of this receptor, its proclivity for heterodimerization, the oddity of EGFR overexpressing EGFR cancers not being slowed down by potent and effective EGFR inhibition, of universal development of resistance to EGFR inhibitors in those cancers initially responsive to EGFR inhibiting drugs, etc.
Line 72. As in comment above re. Line 32, vagueness, “Further the N-glycans of EGFR have been shown to contribute to both the function and regulation of EGFR…”.what does that mean ? you should tell us what - how effect function, what regulation, inhibit, stimulate or what ?
Do the authors think that a Table showing definitions and proliferative effects of BE(2)-C,
BE(2)-C +GnT-I, BE(2)-C(-MGAT1), and BE(2)-C(+/-MGAT1) would be useful ? Glycobiology is important yet somewhat neglected currently. Most readers might benefit from better didactic in your paper.
Line 266, does less processed mean smaller mol. wt. glycans, or fewer in number, or less branched, or what ?
Line 346, please forgive if I am wrong here, but I dont think autophosphorylation is essential for normal or pathological EGFR function. As heterodimer partner to a wide variety of non HER partners - over a dozen are currently recognized - EGFR can become phosphorylated and signaling active when the heterodimer partner has been liganded.
Options consideration: Do the authors think that work on N-glycans in cancer already from 1974 is worth mentioning ? [Oncology. 1974;29:249–264. doi:10.1159/000224907.]
Author Response
Comments to Reviewer 2
Thanks for your comments as the paper has been strengthened especially with regards to the introduction and for a more general audience.
By scoring Novelty and Significance of content as "average" this is not to be construed as a negative. Read "average" as good content worth publishing and reading, but not astounding or paradign shifting.
The subject of N-N-glycosylation is important and currently underappreciated in oncology.
After a necessary rewrite, this paper will be a small but welcome step in the right direction to further awareness of N--glycosylation’s importance in NB and in cancer generally.
Although medical English use is generally good, grammatically and idiomatically correct, this paper requires a comprehensive rewrite. It has not been proofread. There are numerous obviously omitted words, strange sentences, and vagueness. These errors are so numerous that I stopped enumerating them after five such errors in the first 50 lines.
Line 2, title might be better without the word “sensitizes”. Although using “sensitized” is correct as it stands, in current usage sensitized usually refers to increasing effect of a drug. Would “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF Stimulated Proliferation” work better ? Also the title must specify in vitro. So, “Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma in vitro Invasiveness and EGF Stimulated Proliferation”.
Title has been changed.
Line 34. dont be vague, “which is unsatisfactory due to” . Tell us approximately what the median 5 yr OS or some OS range. A 5 year OS of 99% is unsatisfactory too.
The text has been edited to specify the 5 year survival rate in high-risk NB.
Line 36, “of solid NB” ? What other kind of NB is there ? NB is not a leukemia.
The text has been edited to state NB tumors.
Line 38, You are not going to “To improve the quality of NB patients…” They are beautiful, children, equal to any other children.
The text has been edited as follows: “To improve the quality of life for NB patients and avoid side effects from treatment, new therapeutic and diagnostic targets are needed.”
Line 40 should start a new paragraph.
A new paragraph was started with line 40.
Line 49 errors. You are addressing a wide variety of readers, some are clinician-researchers. They will most likely not remember exactly N-glycosylation steps. A figure here is required. Also note that the correct use is N-glycosylation, with lower case g. N-glycans, oligomannose, hybrid, and complex all must be defined in words and shown in a figure. Most of your readers will require reminders here.
Text has been modified to define oligomannose, hybrid, and complex type N-glycans, as well as, referenced Figure 1A. Further we added information to figure and figure legend.
I stopped enumerating errors at line 50. They do, however, continue throughout the manuscript.
Line 62, it might be ok to use EGFR without first spelling it out, it might be preferable though to mention its synonyms HER1 and ErbB1. Any discussion of EGFR must have a paragraph or two giving the broad outline of this receptor, its proclivity for heterodimerization, the oddity of EGFR overexpressing EGFR cancers not being slowed down by potent and effective EGFR inhibition, of universal development of resistance to EGFR inhibitors in those cancers initially responsive to EGFR inhibiting drugs, etc.
Text has been modified to include an additional paragraph outlining EGFR. Also, original text has been reorganized to provide clarity on the glycosylation of EGFR.
Line 72. As in comment above re. Line 32, vagueness, “Further the N-glycans of EGFR have been shown to contribute to both the function and regulation of EGFR…”.what does that mean ? you should tell us what - how effect function, what regulation, inhibit, stimulate or what ?
The following text has been added for clarity:
“Changes in the N-glycan processing of EGFR can promote or diminish EGFR signaling, with complete inhibition of N-glycosylation greatly reducing EGFR signaling (29-33).”
Do the authors think that a Table showing definitions and proliferative effects of BE(2)-C,
BE(2)-C +GnT-I, BE(2)-C(-MGAT1), and BE(2)-C(+/-MGAT1) would be useful ? Glycobiology is important yet somewhat neglected currently. Most readers might benefit from better didactic in your paper.
Figure 1A has been updated and a more thorough description added in paragraph 3 of introduction.
Line 266, does less processed mean smaller mol. wt. glycans, or fewer in number, or less branched, or what ?
Sentence has been changed to: “However, the electrophoretic migration of EGFR was noticeably faster in the mutant cell line, indicating that the N-glycans of EGFR are composed of reduced monosaccharides and antenna, thus suggesting less processed N-glycans (i.e. oligomannose type N-glycans) than in the parental cell line.”
Line 346, please forgive if I am wrong here, but I dont think autophosphorylation is essential for normal or pathological EGFR function. As heterodimer partner to a wide variety of non HER partners - over a dozen are currently recognized - EGFR can become phosphorylated and signaling active when the heterodimer partner has been liganded.
Although we recognize exceptions may occur, here we are describing EGFR signaling following EGF stimulation which does result in autophosphorylation.
Options consideration: Do the authors think that work on N-glycans in cancer already from 1974 is worth mentioning ? [Oncology. 1974;29:249–264. doi:10.1159/000224907.]
We did not reference as it does not deal with NB.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have modified the manuscript in accordance with my objections. For me, the manuscript is now publishable in the journal
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Many thanks for your comments as they improved the manuscript.
Reviewer 2 Report
Comments and Suggestions for Authors
Important findings are reported in this paper showing in vitro effects of glycosylation effects on EGFR and cell migration/growth. These finding deepen our understanding of EGFR. Exposition was still poor, but maybe not poor enough to exclude publication. General English language editing will not help much - the language editor must be a glycosylation expert, which I am not.
I find that this report was difficult to read and understand fully. Like many readers of EGFR data, I am not well versed in glycosylation effects. I agree with the authors’ comment that glycosylation is “vastly understudied”. So a better introduction will help the non-expert understand this work.
Paying more attention to the didactic function of your paper would help redressing that. Add a Table with glycosylation related terms ? Or a glossary Figure ?
Also better to delete emotive terms like “vastly”.
Again I would complain about vagueness at many places throughout the manuscript. Can you correct this imprecise language use ? Be specific. Can you avoid words like “modulate”, “aberrant”, line 41 “disruptions”,
Line 43, make it clear that reduced glycosylation increases function of other receptors too. Do we have examples of reduced receptor function when glycosylation is full or overfull ? If yes, then disruption or aberrant would be correct words to use but then would require explaining both conditions explicitly and with examples.
Lines 42, 42 you correctly use specific terms “reduced occupancy” and “reduction of N-glycans with polysialic acid associated with neural cell adhesion molecule (NCAM)”
Figure 1 on page 3 and the cartoon on page 7 are the same ?
Line 58. A Table summarizing this would help the general medical reader.
Line 85. “Processing” can mean adding or subtracting.
Line 102. Do we know if a heterodimer EGFR-XYZ, will be created and activated by either ligand [EGF or XYZ or both ] ? it is a bit tangential but
Line 112. This is a strong point, emphasize it - Nonglycosylated EGFR functions poorly as a receptor.
Line 128. The go-or-grow phenomenon is interesting. Your comment “significant role of oligomannose N-glycans in decreasing cell proliferation and increasing invasion in human and rat NB” implies an intracellular switching process. Another physiological response is the migration enhancing effect of cytotoxic meds or radiation. So you have identified a signpost or indicator that switching process. We would like to know if the glycosylation process you mention, oligomannose N-glycans, is a link mediating the switch from growth to migration, or a link in the signaling process causing the switch. Can you contribute to this aspect ? Please forgive me if I have misunderstood something here.
Line 524. Re “The high levels of oligomannose N-glycans reduced cell proliferation using standard 2D and 3D cell cultures; however, they greatly increased cell invasiveness using 3D cell cultures.” is important. Since we want neither migration nor growth, what glycosylation state might contribute to that ?
Re. EGFR itself, we see in other cancers that even deep inhibition of all EGFR signaling does not cure - alternate signaling eventually cross covers.
Line 561. Did I understand correctly that you are suggesting that over oligomannosylated of EGFR is one of the causes of constitutively activated EGFR ? If yes, say so.
Line 574. Do not say “changed”. State specifically what you intend - increased or decreased hydrophilicity.
Line 630. How, by what mechanism, does glycosylation mediate difference between 2D cell culture and 3D cell culture ?
Important findings are reported in this paper showing in vitro effects of glycosylation effects on EGFR and cell migration/growth. These finding deepen our understanding of EGFR. Exposition was still poor, but maybe not poor enough to exclude publication. General English language editing will not help much - the language editor must be a glycosylation expert, which I am not.
I find that this report was difficult to read and understand fully. Like many readers of EGFR data, I am not well versed in glycosylation effects. I agree with the authors’ comment that glycosylation is “vastly understudied”. So a better introduction will help the non-expert understand this work.
Paying more attention to the didactic function of your paper would help redressing that. Add a Table with glycosylation related terms ? Or a glossary Figure ?
Also better to delete emotive terms like “vastly”.
Again I would complain about vagueness at many places throughout the manuscript. Can you correct this imprecise language use ? Be specific. Can you avoid words like “modulate”, “aberrant”, line 41 “disruptions”,
Line 43, make it clear that reduced glycosylation increases function of other receptors too. Do we have examples of reduced receptor function when glycosylation is full or overfull ? If yes, then disruption or aberrant would be correct words to use but then would require explaining both conditions explicitly and with examples.
Lines 42, 42 you correctly use specific terms “reduced occupancy” and “reduction of N-glycans with polysialic acid associated with neural cell adhesion molecule (NCAM)”
Figure 1 on page 3 and the cartoon on page 7 are the same ?
Line 58. A Table summarizing this would help the general medical reader.
Line 85. “Processing” can mean adding or subtracting.
Line 102. Do we know if a heterodimer EGFR-XYZ, will be created and activated by either ligand [EGF or XYZ or both ] ? it is a bit tangential but
Line 112. This is a strong point, emphasize it - Nonglycosylated EGFR functions poorly as a receptor.
Line 128. The go-or-grow phenomenon is interesting. Your comment “significant role of oligomannose N-glycans in decreasing cell proliferation and increasing invasion in human and rat NB” implies an intracellular switching process. Another physiological response is the migration enhancing effect of cytotoxic meds or radiation. So you have identified a signpost or indicator that switching process. We would like to know if the glycosylation process you mention, oligomannose N-glycans, is a link mediating the switch from growth to migration, or a link in the signaling process causing the switch. Can you contribute to this aspect ? Please forgive me if I have misunderstood something here.
Line 524. Re “The high levels of oligomannose N-glycans reduced cell proliferation using standard 2D and 3D cell cultures; however, they greatly increased cell invasiveness using 3D cell cultures.” is important. Since we want neither migration nor growth, what glycosylation state might contribute to that ?
Re. EGFR itself, we see in other cancers that even deep inhibition of all EGFR signaling does not cure - alternate signaling eventually cross covers.
Line 561. Did I understand correctly that you are suggesting that over oligomannosylated of EGFR is one of the causes of constitutively activated EGFR ? If yes, say so.
Line 574. Do not say “changed”. State specifically what you intend - increased or decreased hydrophilicity.
Line 630. How, by what mechanism, does glycosylation mediate difference between 2D cell culture and 3D cell culture ?
Author Response
Point-by-point comments to reviewer 2
Authors: We are thankful for the time you spent reviewing our manuscript as the quality has been improved.
Important findings are reported in this paper showing in vitro effects of glycosylation effects on EGFR and cell migration/growth. These finding deepen our understanding of EGFR. Exposition was still poor, but maybe not poor enough to exclude publication. General English language editing will not help much - the language editor must be a glycosylation expert, which I am not.
I find that this report was difficult to read and understand fully. Like many readers of EGFR data, I am not well versed in glycosylation effects. I agree with the authors’ comment that glycosylation is “vastly understudied”. So a better introduction will help the non-expert understand this work.
Paying more attention to the didactic function of your paper would help redressing that. Add a Table with glycosylation related terms ? Or a glossary Figure ?
Authors: We have changed figure 1 to enhance the understanding of readers not well-versed in glycosylation.
Also better to delete emotive terms like “vastly”.
Authors: removed “vastly”
Again I would complain about vagueness at many places throughout the manuscript. Can you correct this imprecise language use ? Be specific. Can you avoid words like “modulate”, “aberrant”, line 41 “disruptions”,
Authors: This has been reviewed and edited as needed.
Line 43, make it clear that reduced glycosylation increases function of other receptors too. Do we have examples of reduced receptor function when glycosylation is full or overfull ? If yes, then disruption or aberrant would be correct words to use but then would require explaining both conditions explicitly and with examples.
Authors: yes, ref 14 is vacancy of N-glycosylation sites while ref 15 deals with reduction of polysialylated N-glycans which is altered processing the N-glycan when it is attached to protein.
Lines 42, 42 you correctly use specific terms “reduced occupancy” and “reduction of N-glycans with polysialic acid associated with neural cell adhesion molecule (NCAM)”
Authors: Okay. See lines 90-92 to clearly define occupancy and altered processed N-glycans associated with glycoproteins.
Figure 1 on page 3 and the cartoon on page 7 are the same ?
Authors: It is unclear as there is not a figure 1 is not duplicated on our copy. However, as mentioned above figure 1 has been revised to enhance the understanding of our results by the reader.
Line 58. A Table summarizing this would help the general medical reader.
Authors: This has been accomplished, see revised figure 1 and legend.
Line 85. “Processing” can mean adding or subtracting.
Authors: Correct
Line 102. Do we know if a heterodimer EGFR-XYZ, will be created and activated by either ligand [EGF or XYZ or both ] ? it is a bit tangential but
Authors: It is unclear how heterodimerization and ligand binding would be impacted but would be interested to pursue this in future studies.
Line 112. This is a strong point, emphasize it - Nonglycosylated EGFR functions poorly as a receptor.
Authors: The sentence has been modified.
Line 128. The go-or-grow phenomenon is interesting. Your comment “significant role of oligomannose N-glycans in decreasing cell proliferation and increasing invasion in human and rat NB” implies an intracellular switching process. Another physiological response is the migration enhancing effect of cytotoxic meds or radiation. So you have identified a signpost or indicator that switching process. We would like to know if the glycosylation process you mention, oligomannose N-glycans, is a link mediating the switch from growth to migration, or a link in the signaling process causing the switch. Can you contribute to this aspect ? Please forgive me if I have misunderstood something here.
Authors: Our 3D cell spheroid data suggest that when complex N-glycans are replaced by oligomannose N-glycan they will produce cells that are more invasive (Hall et al., 2021; 2023), and that these cells have a greater response to EGFR by proliferating at a faster rate (shown in current paper). Thus, it appears that expression of specific types of N-glycans are acting as a switch.
Line 524. Re “The high levels of oligomannose N-glycans reduced cell proliferation using standard 2D and 3D cell cultures; however, they greatly increased cell invasiveness using 3D cell cultures.” is important. Since we want neither migration nor growth, what glycosylation state might contribute to that ?
Authors: As mentioned, our 3D cell spheroid data suggest that when complex N-glycans are replaced by oligomannose N-glycan they will produce cells that are more invasive but less proliferative (Hall et al., 2021; 2023). However, proliferation is greatly increased in response to EGFR when cells express high levels of oligomannose (shown in current paper). Therefore, oligomannose N-glycans greatly contribute to promoting invasiveness and proliferation.
Re. EGFR itself, we see in other cancers that even deep inhibition of all EGFR signaling does not cure - alternate signaling eventually cross covers.
Authors: Based on our results, the cells expressing high levels of oligomannose would still be highly invasive.
Line 561. Did I understand correctly that you are suggesting that over oligomannosylated of EGFR is one of the causes of constitutively activated EGFR ? If yes, say so.
Authors: Correct, “The scenario supported by our results would be that the 3-dimensional structure of oligomannosylated EGFR would be more inclined to dimerize, have increased autophosphorylation without or with EGF stimulation, and more prone to undergo EGF stimulated proliferation”
Line 574. Do not say “changed”. State specifically what you intend - increased or decreased hydrophilicity.
Authors: This has been modified. “decrease the hydrophilicity and increase dynamics”
Line 630. How, by what mechanism, does glycosylation mediate difference between 2D cell culture and 3D cell culture ?
Authors: This is an ongoing area of research. Higher expression of complex N-glycans, higher expression of oligomannose N-glycans, and no changes have all been reported in various cell lines when cultured in 3D compared to 2D. In our NB cells lines, we observed higher oligomannose N-glycan levels using 3D cultures (personal observation). It is likely that the culturing method impacts the N-glycosylation pathway. A likely factor would be cell-cell contacts (e.g., glycosynapses); however, this is not completely understood and is an area of ongoing research.
Author Response File: Author Response.pdf
