The Specific Role of Dermatan Sulfate as an Instructive Glycosaminoglycan in Tissue Development

Round 1

Reviewer 1 Report

I find the paper well written and comprehensive. I have two suggestions. Table 2 should be referenced properly in the Table heading.

The second suggestion concerns section 3 which discusses the knockout mice. It is not clear that the work was performed by others even though the reference was placed near the end of the paragraph. In a review article, it is always preferable to show respect for the scientists by beginning the sentence, as in line 148 as follows, or similar:

In 2009, Maccarana and coworkers generated DSE knock-out mice. Their studies showed that….

It would be more respectful to treat each knock out mouse using the years and names of the scientists.

Otherwise, the paper is well done

Author Response

Comments and Suggestions for Authors from Reviewer 1

I find the paper well written and comprehensive. I have two suggestions.

Table 2 should be referenced properly in the Table heading.

The second suggestion concerns section 3 which discusses the knockout mice. It is not clear that the work was performed by others even though the reference was placed near the end of the paragraph. In a review article, it is always preferable to show respect for the scientists by beginning the sentence, as in line 148 as follows, or similar:

In 2009, Maccarana and coworkers generated DSE knock-out mice. Their studies showed that….  It would be more respectful to treat each knock out mouse using the years and names of the scientists.

Otherwise, the paper is well done

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, we have cited the references in Table 1 (the former Table 2) (page5).

   To show respect for the scientists by beginning the sentence, the years and names of the scientists have been described in each knock-out mice (Lines 15-154, 162, 180-181, 196-197, 210-211, 219, 227, 232, and 241).

Comments and Suggestions for Authors from Reviewer 2

The authors present a comprehensive study of up-to-date research data-base. upon a field of interest such as the role of dermatan sulphate in tissue development. The review comprehends a possible role of dermatan sulphate in such a role. The introduction and biosynthesis of chains dermatan sulphate are relevant and clearly described. Illustrations and Tables are relevant as well as conclusions.

Our response:

   We thank the reviewer’s comment.

Comments and Suggestions for Authors from Reviewer 3

This review features discussions on the roles of dermatan sulfate (DS) in tissue development. DS chains consist of disaccharide units constructed by specific glycosyltransferases and epimerase. They summarized knockout and mutant mice of biosynthetic enzymes of DS, such as Dse, Dsel, and Chst14. Mice deficient in DS demonstrated replacement with chondroitin sulfate and anomalies of the skin, skeleton, nervous system, and early development. The authors predict that DS-deficient model mice are potent tools for developing new therapeutics for Ehlers-Danlos syndrome caused by DS defects. The following points should be clarified.

1. Table 1 is missing.
2. The references are missing in table 2.
3. Page 7, line 288: anormalies→anomalies (?)

Our response:

   We thank the reviewer for this valuable suggestion.

1) Table 2 was corrected into Table 1 (page 5).

2) we have cited the references in Table 1 (the former Table 2) (page 5).

3) It has been corrected.

Additional corrections:

1) The following sentence has been added in the Introduction section, lines 30-32.

“DS-PGs play important roles in anti-coagulation, binding to growth factors, wound healing, assembly of extracellular matrices, tissue morphogenesis, and neuronal homeostasis [3-9].”

2) The references have been renumbered due to being cited new references (Refs. #7-9).

3) Table 2 - - - > Table 1

4) IdoUA, GlcUA - - - > IdoA. GlcA (lines 37, 40)

Reviewer 2 Report

The authors present a comprehensive study of up-to-date research data-base. upon a field of interest such as the role of dermatan sulphate in tissue development. The review comprehends a possible role of dermatan sulphate in such a role. The introduction and biosynthesis of chains dermatan sulphate are relevant and clearly described. Illustrations and Tables are relevant as well as conclusions. 

Author Response

Comments and Suggestions for Authors from Reviewer 1

I find the paper well written and comprehensive. I have two suggestions.

Table 2 should be referenced properly in the Table heading.

The second suggestion concerns section 3 which discusses the knockout mice. It is not clear that the work was performed by others even though the reference was placed near the end of the paragraph. In a review article, it is always preferable to show respect for the scientists by beginning the sentence, as in line 148 as follows, or similar:

In 2009, Maccarana and coworkers generated DSE knock-out mice. Their studies showed that….  It would be more respectful to treat each knock out mouse using the years and names of the scientists.

Otherwise, the paper is well done

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, we have cited the references in Table 1 (the former Table 2) (page5).

   To show respect for the scientists by beginning the sentence, the years and names of the scientists have been described in each knock-out mice (Lines 15-154, 162, 180-181, 196-197, 210-211, 219, 227, 232, and 241).

Comments and Suggestions for Authors from Reviewer 2

The authors present a comprehensive study of up-to-date research data-base. upon a field of interest such as the role of dermatan sulphate in tissue development. The review comprehends a possible role of dermatan sulphate in such a role. The introduction and biosynthesis of chains dermatan sulphate are relevant and clearly described. Illustrations and Tables are relevant as well as conclusions.

Our response:

   We thank the reviewer’s comment.

Comments and Suggestions for Authors from Reviewer 3

This review features discussions on the roles of dermatan sulfate (DS) in tissue development. DS chains consist of disaccharide units constructed by specific glycosyltransferases and epimerase. They summarized knockout and mutant mice of biosynthetic enzymes of DS, such as Dse, Dsel, and Chst14. Mice deficient in DS demonstrated replacement with chondroitin sulfate and anomalies of the skin, skeleton, nervous system, and early development. The authors predict that DS-deficient model mice are potent tools for developing new therapeutics for Ehlers-Danlos syndrome caused by DS defects. The following points should be clarified.

1. Table 1 is missing.
2. The references are missing in table 2.
3. Page 7, line 288: anormalies→anomalies (?)

Our response:

   We thank the reviewer for this valuable suggestion.

1) Table 2 was corrected into Table 1 (page 5).

2) we have cited the references in Table 1 (the former Table 2) (page 5).

3) It has been corrected.

Additional corrections:

1) The following sentence has been added in the Introduction section, lines 30-32.

“DS-PGs play important roles in anti-coagulation, binding to growth factors, wound healing, assembly of extracellular matrices, tissue morphogenesis, and neuronal homeostasis [3-9].”

2) The references have been renumbered due to being cited new references (Refs. #7-9).

3) Table 2 - - - > Table 1

4) IdoUA, GlcUA - - - > IdoA. GlcA (lines 37, 40)

Reviewer 3 Report

The manuscript “The specific role of dermatan sulfate as an instructive glycosaminoglycan in tissue development” of Mizumoto and Yamada, is a review paper that aims to elucidate the role of the dermatan sulfate in tissue development mainly through data reported in literature from experiments on mice and from clinical data on human studies. The paper is interesting and suitable for the journal, but it could not be published before performing some minor revisions.

Here some specific comments.

-Please clarify in the abstract and in the introduction that this paper is a review and clearly indicate on which period it is focused on. The last 10 years? 15 years? It is not clearly indicated, and my suggestion is to remove from this review any papers older than 15 years.

-All the data reported in the paper on the CS/DS ratio are mentioned without saying which methods have been used to determine the difference between the IdoA and GlcA contents in disaccharide composition or to differentiate DS from CS. The length of the chains and the grade of sulfation are also critical for DS biological activity (as reported in lines 112-120). Thus please specify it in the paper and eventually put a small table on the methods used to get all these data, eventually also comparing these methods with the ones commonly used to characterize the CS (See Restaino, O.F., Schiraldi, C. Carbohydrate Polymers, 2022 and Restaino, O.F. et al., Analytica Chimica Acta, 2017).

-Paragraph 2: If the 4 and the 2 sulfation is predominant in DS, as said in the text, please specify in which percentages and how low is the 6 sulfation in comparison to CS (See Restaino, O.F. et al., Analytica Chimica Acta, 2017).

-line 82: please here indicate that 3’-phosphoadenosine 5’-phosphosulfate is PAPS as this acronyms is then used in the paper.

-Paragraph 3: Which are the classical functions of DS? Please specify them.

-Paragraph 3: Is the mice the only animal model employed so far for studying the DS enzyme alteration? Are there other animal models? If yes, please insert in the review all of the other models reported in literature.

-Paragraph 3: According to me it could be useful to make a comparison and to insert in Fig 2 a parallel with the alteration of the genes causing changing in CS structure and the consequences on the normal physiology of tissues and organs as here reported for DS.

-Paragraph 4: Are there already protocols used in clinical studies to counteract the alteration of DS enzyme functions, as in mucopolysaccharidosis, as for example by using a replacement of enzymes by exogenous administration? Insert these data in the review too.

-Which is the life expectation of patients having a DS enzyme genetic disorder? Insert these data in the review.

-The relation between the DS structure alteration and cancer is not well described in this review. Please insert a paragraph on the new results on the involvement of the DS enzymes in the proliferation of the different types of cancer. Do the metastatic cancer forms depend on an alteration of the matrix composition in terms of more or less DS presence? In terms of higher or lower CS/DS disaccharide ratio in the synthesis of GAG chains? In terms of higher or lower GlcA/IdoA disaccharide ratio and more or less sulfated disaccharides synthesis?

-Figure 1: In the parentheses describing the DS disaccharide the 6 sulfation is indicated as possible, but it is not indicated in the picture of the disaccharide structure. Please indicate it and specify better the percentages of 4, 2 and 6 sulfation in DS compared to the CS structure.

-Figure 2: Please specify here in the legend and in the text that Chn is the unsulfate backbone of CS.

Author Response

Comments and Suggestions for Authors from Reviewer 3

The manuscript “The specific role of dermatan sulfate as an instructive glycosaminoglycan in tissue development” of Mizumoto and Yamada, is a review paper that aims to elucidate the role of the dermatan sulfate in tissue development mainly through data reported in literature from experiments on mice and from clinical data on human studies. The paper is interesting and suitable for the journal, but it could not be published before performing some minor revisions.

Here some specific comments.

1) -Please clarify in the abstract and in the introduction that this paper is a review and clearly indicate on which period it is focused on. The last 10 years? 15 years? It is not clearly indicated, and my suggestion is to remove from this review any papers older than 15 years.

Our response:

   We thank the reviewer for this valuable suggestion. We mainly focused on the functions of DS based on findings of the last decade using knock-out mice and human disorders. The following word “in the past decay” has been described in the Abstract and Introduction sections.

“This review highlights a novel role of DS in tissue development in the past decade.” (lines 20-21, Abstract)

“This review focuses and discusses on not only functions of DS but also DS-defective model animals in the past decade.” (lines 70-71, Introduction)

The reviewer’s comment for cited papers is reasonable. However, the authors feel that the original papers and important review articles should be cited even though old papers. Because this review includes the classical functions of DS, which have been demonstrated more than 20-years ago, to briefly introduce to the audiences. Thus, the authors did not correct the cited papers.

2) -All the data reported in the paper on the CS/DS ratio are mentioned without saying which methods have been used to determine the difference between the IdoA and GlcA contents in disaccharide composition or to differentiate DS from CS. The length of the chains and the grade of sulfation are also critical for DS biological activity (as reported in lines 112-120). Thus please specify it in the paper and eventually put a small table on the methods used to get all these data, eventually also comparing these methods with the ones commonly used to characterize the CS (See Restaino, O.F., Schiraldi, C. Carbohydrate Polymers, 2022 and Restaino, O.F. et al., Analytica Chimica Acta, 2017).

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, Table 1 has been added in the manuscript as follows.

Table 1. Biological activity of a variety of DS variant.

––, not reported

3) Paragraph 2: If the 4 and the 2 sulfation is predominant in DS, as said in the text, please specify in which percentages and how low is the 6 sulfation in comparison to CS (See Restaino, O.F. et al., Analytica Chimica Acta, 2017).

Our response:

   In the paragraph 2, it was described that “The DS-derived hexasaccharide, [-IdoA(2-O-sulfate)-GalNAc(4-O-sulfate)-]3, from porcine skin has been identified as the smallest fragment of DS binding to heparin cofactor II with high affinity [51].” The hexasaccharide no contains 6-O-sulfation.

4) line 82: please here indicate that 3’-phosphoadenosine 5’-phosphosulfate is PAPS as this acronyms is then used in the paper.

Our response:

   As suggested by the reviewer, the “PAPS” has been utilized in the manuscript.

5) Paragraph 3: Which are the classical functions of DS? Please specify them.

Our response:

   We thank the reviewer for this valuable suggestion. As pointed-out by the reviewer, the paragraph has been divided into two sub-paragraphs, “3.1. Classical functions of DS” and “3.2. Recent additional functions of DS”.

6) Paragraph 3: Is the mice the only animal model employed so far for studying the DS enzyme alteration? Are there other animal models? If yes, please insert in the review all of the other models reported in literature.

Our response:

   We thank the reviewer for this valuable suggestion. Knockdown of D4ST1 in zebrafish has been reported. Thus, the phenotypes of the D4ST1-morphants have been described in the Section 3.2 as follows.

“Although Knockdown of C4ST1 by antisense morpholino oligonucleotide accelerated regeneration of after spinal cord injury in zebrafish, knockdown of D4ST1 did not [67], indicating that 4-O-sulfation of CS, but not DS, inhibit axonal regrowth after spinal cord injury.”

7) Paragraph 3: According to me it could be useful to make a comparison and to insert in Fig 2 a parallel with the alteration of the genes causing changing in CS structure and the consequences on the normal physiology of tissues and organs as here reported for DS.

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, new Table 3 has been described in the text as described below.

Table 3. Alteration of CS/DS disaccharides in the skin fibroblasts from patients with mutations in DSE and CHST14.

*compared with the healthy subjects

8) Paragraph 4: Are there already protocols used in clinical studies to counteract the alteration of DS enzyme functions, as in mucopolysaccharidosis, as for example by using a replacement of enzymes by exogenous administration? Insert these data in the review too.

Our response:

   We thank the reviewer for this suggestion. However, an enzyme replacement therapy for mucopolysaccharidosis out of the main subject of this review. Thus, it has not been described.

9) Which is the life expectation of patients having a DS enzyme genetic disorder? Insert these data in the review.

Our response:

   We thank the reviewer for this suggestion. However, it remains unclear so far. Thus, there was no description of the life expectation of patients with mutations in DSE and CHST14.

10) The relation between the DS structure alteration and cancer is not well described in this review. Please insert a paragraph on the new results on the involvement of the DS enzymes in the proliferation of the different types of cancer. Do the metastatic cancer forms depend on an alteration of the matrix composition in terms of more or less DS presence? In terms of higher or lower CS/DS disaccharide ratio in the synthesis of GAG chains? In terms of higher or lower GlcA/IdoA disaccharide ratio and more or less sulfated disaccharides synthesis?

Our response:

   We thank the reviewer for this valuable suggestion. The following sentences have been described in the text.

“Furthermore, IdoA-deficient human esophagus squamous cell carcinoma by shRNA showed decreased migration and invasion capabilities in vitro, which was associated with reduced cellular interaction with hepatocyte growth factor, inhibition of pERK-1/2 sig-naling, and deregulated actin cytoskeleton dynamics and focal adhesion formation [64].” (lines 136-140)

“However, it remains unclear the ratio of CS/DS, content of IdoA, chain length, sulfation pattern, binding molecules, and cell signaling.” (lines 142-3)

11) Figure 1: In the parentheses describing the DS disaccharide the 6 sulfation is indicated as possible, but it is not indicated in the picture of the disaccharide structure. Please indicate it and specify better the percentages of 4, 2 and 6 sulfation in DS compared to the CS structure.

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, the 6-O-sulfation in DS has been described in Fig. 1.

The references for percentage of disaccharide units in CS and DS have been cited in the legend.

12) Figure 2: Please specify here in the legend and in the text that Chn is the unsulfate backbone of CS.

Our response:

   We thank the reviewer for this suggestion. As suggested, the following sentence has been described in the text and the legend of Fig. 2.

“which is the unsulfate backbone of CS”

Author Response File: Author Response.pdf

Reviewer 4 Report

This review features discussions on the roles of dermatan sulfate (DS) in tissue development. DS chains consist of disaccharide units constructed by specific glycosyltransferases and epimerase. They summarized knockout and mutant mice of biosynthetic enzymes of DS, such as Dse, Dsel, and Chst14. Mice deficient in DS demonstrated replacement with chondroitin sulfate and anomalies of the skin, skeleton, nervous system, and early development. The authors predict that DS-deficient model mice are potent tools for developing new therapeutics for Ehlers-Danlos syndrome caused by DS defects. The following points should be clarified.

1.       Table 1 is missing.

2.       The references are missing in table 2.

3.  Page 7, line 288: anormalies→anomalies (?)

Author Response

Comments and Suggestions for Authors from Reviewer 1

I find the paper well written and comprehensive. I have two suggestions.

Table 2 should be referenced properly in the Table heading.

The second suggestion concerns section 3 which discusses the knockout mice. It is not clear that the work was performed by others even though the reference was placed near the end of the paragraph. In a review article, it is always preferable to show respect for the scientists by beginning the sentence, as in line 148 as follows, or similar:

In 2009, Maccarana and coworkers generated DSE knock-out mice. Their studies showed that….  It would be more respectful to treat each knock out mouse using the years and names of the scientists.

Otherwise, the paper is well done

Our response:

   We thank the reviewer for this valuable suggestion. As suggested, we have cited the references in Table 1 (the former Table 2) (page5).

   To show respect for the scientists by beginning the sentence, the years and names of the scientists have been described in each knock-out mice (Lines 15-154, 162, 180-181, 196-197, 210-211, 219, 227, 232, and 241).

Comments and Suggestions for Authors from Reviewer 2

The authors present a comprehensive study of up-to-date research data-base. upon a field of interest such as the role of dermatan sulphate in tissue development. The review comprehends a possible role of dermatan sulphate in such a role. The introduction and biosynthesis of chains dermatan sulphate are relevant and clearly described. Illustrations and Tables are relevant as well as conclusions.

Our response:

   We thank the reviewer’s comment.

Comments and Suggestions for Authors from Reviewer 3

This review features discussions on the roles of dermatan sulfate (DS) in tissue development. DS chains consist of disaccharide units constructed by specific glycosyltransferases and epimerase. They summarized knockout and mutant mice of biosynthetic enzymes of DS, such as Dse, Dsel, and Chst14. Mice deficient in DS demonstrated replacement with chondroitin sulfate and anomalies of the skin, skeleton, nervous system, and early development. The authors predict that DS-deficient model mice are potent tools for developing new therapeutics for Ehlers-Danlos syndrome caused by DS defects. The following points should be clarified.

1. Table 1 is missing.
2. The references are missing in table 2.
3. Page 7, line 288: anormalies→anomalies (?)

Our response:

   We thank the reviewer for this valuable suggestion.

1) Table 2 was corrected into Table 1 (page 5).

2) we have cited the references in Table 1 (the former Table 2) (page 5).

3) It has been corrected.

Additional corrections:

1) The following sentence has been added in the Introduction section, lines 30-32.

“DS-PGs play important roles in anti-coagulation, binding to growth factors, wound healing, assembly of extracellular matrices, tissue morphogenesis, and neuronal homeostasis [3-9].”

2) The references have been renumbered due to being cited new references (Refs. #7-9).

3) Table 2 - - - > Table 1

4) IdoUA, GlcUA - - - > IdoA. GlcA (lines 37, 40)

Round 2

Reviewer 4 Report

The authors addressed the points which I noted.

Author Response

Thank you for your comment.