Review Reports - EAE of Mice: Enzymatic Cross Site-Specific Hydrolysis of H2B Histone by IgGs against H1, H2A, H2B, H3, and H4 Histones and Myelin Basic Protein

Round 1

Reviewer 1 Report

Urusov et al. analyzed the H2B hydrolysis products using abzymes against different histones or myelin basic protein, and they found that there is a cross-catalytic activity of the IgG. Moreover, they claimed that histone/MOG triggered IgGs have different numbers and/or different cleavage sites, suggesting these differences might be related to the EAE development. However, all these experiments are done with EAE mouse models. While biochemically it’s hard to imagine or prove how it works in the experiment setup in this manuscript. But technically, it would be interesting to test this observation in a healthy mouse for comparison. Overall, I am feeling this study lacks scientific soundness, and I don’t feel the histone hydrolysis pattern has anything to do with EAE development in mice.

I might miss the point somewhere in the manuscript, did you use specific histone antibody to purify the IgG so that you start with an ideally monoclonal IgG to test the catalytic activity toward H2B histone? Because you used auto-immune-diseased mice, there might already abzymes in mice without treatment of histone or histone/DNA complexes. That could explain the control sample that also shows catalytic activity. Another question related to this is that when you purify the IgG from those control mice samples, did you get similar yield of IgG compared to the ones from mice that are treated with specific histones. There is a chance that it already has a mixture of abzymes existing in the mice, that show diverse H2B cleavage. This observation was in consistent with your experimental data, all the controls have different H2B cleavage sites, and there is no statistically reliable pattern about the H2B cleavage sites in different scenarios, including controls or mices treated with histone/DNA complexes/MOG.

Minor points:

Lots of acronyms without specifying them. For example, I don’t know what MOG is. Please specify all the acronyms at their first instances.

References needed for the statements (lines 49-53)

For Fig1, I would like to see the full gel pictures especially in the supplementary gel/blots file, not just the bands. I don’t know what’s going on with Fig1 panel B, and I believe that is histone H1.

In Fig6, is the data for H2A or H2B, very confusing.

Could you put Fig7-11 side by side to Fig 2-6, respectively, for easy comparison?

In line 517, could you specify what’s the sequence identity between all histones? Especially H1 is definitely not sharing high homology with H2A/H2B, so I would be very cautious about your explanation.

Author Response

Urusov et al. analyzed the H2B hydrolysis products using abzymes against different histones or myelin basic protein, and they found that there is a cross-catalytic activity of the IgG. Moreover, they claimed that histone/MOG triggered IgGs have different numbers and/or different cleavage sites, suggesting these differences might be related to the EAE development. However, all these experiments are done with EAE mouse models. While biochemically it’s hard to imagine or prove how it works in the experiment setup in this manuscript. But technically, it would be interesting to test this observation in a healthy mouse for comparison. Overall, I am feeling this study lacks scientific soundness, and I don’t feel the histone hydrolysis pattern has anything to do with EAE development in mice.

Answer. There are no catalytically active antibodies hydrolyzing histone H2B or other histones in the blood of healthy СBA and other mice not prone to AID [12-23]. After immunization of these mice with MOG, there is no change in the differentiation profile of bone marrow stem cells and the production of abzymes against histones, but only against MOG with very, very weak activity. One of the main disorders in MS patients leading to the disease is the production of antibodies against nerve tissue sheath proteins. Antibodies against histones and MBP, as already shown in a number of works [12-23] and in this study, have cross-catalytic activity. At the same time, as shown earlier using monoclonal antibodies against MBP and DNA, approximately 30-40% of all produced antibodies to one antigen have catalytic activity [68-73]. From the existence of cross-catalytic activity of antibodies against histones and MBP, it follows that anti-histone abzymes can hydrolyze MBP of nerve tissue membranes, which will lead to disruption of the passage of nerve impulses through nerve tissues, which is observed in multiple sclerosis.

It can be allowed that the production of abzymes against histones in MS patients is not directly related to the development of the disease. However, from the entire set of data on abzymes obtained in our laboratory, it follows that the entire set of abzymes against DNA, RNA, histones. MBP, oligo- and polysaccharides destroy the components of blood and cells of different tissues [18-23]. Antibodies against histones, DNA and histone-DNA complexes penetrate into the nuclei of cells and stimulate their apoptosis. The appearance of histone complexes with DNA in the blood as a result of apoptosis leads to an increase in the concentration of antibodies against them, and as a result to an increase in the level of cell apoptosis and incitement of the development of pathology. We came to the conclusion about the role of histones and their complexes in the development of EAE based on the totality of data on such abzymes of patients with MS and EAE mice [18-23].

I might miss the point somewhere in the manuscript, did you use specific histone antibody to purify the IgG so that you start with an ideally monoclonal IgG to test the catalytic activity toward H2B histone? Because you used auto-immune-diseased mice, there might already abzymes in mice without treatment of histone or histone/DNA complexes. That could explain the control sample that also shows catalytic activity. Another question related to this is that when you purify the IgG from those control mice samples, did you get similar yield of IgG compared to the ones from mice that are treated with specific histones. There is a chance that it already has a mixture of abzymes existing in the mice, that show diverse H2B cleavage. This observation was in consistent with your experimental data, all the controls have different H2B cleavage sites, and there is no statistically reliable pattern about the H2B cleavage sites in different scenarios, including controls or mice treated with histone/DNA complexes/MOG.

Answer:

Answer: Sorry, but obtaining monoclonal antibodies from the blood of mice are simply not possible, since from a theoretical point of view, about a million antibodies with different properties can be produced for each antigen. According to our estimates using phage display technology, the blood of SLE patients contains more than a thousand antibodies-abzymes against DNA and against MBP, which differ greatly in their catalytic properties. With this in mind, we isolated the entire population of polyclonal antibodies against five histones using Sepharose containing five immobilized histones. Then, to remove possible admixtures of antibodies against MBP, this preparation was passed through Sepharose with immobilized MBP and collected as a pool of antibodies against five histones that did not bind to this sorbent, released during application. To obtain antibodies against individual histones, we performed affinity chromatography of this preparation on sorbents containing immobilized individual antibodies - H1, H2A, H2B, H3 and H4. Thus, not monoclonal, but polyclonal antibodies were obtained against each of these individual histones, which may contain a set of a large number of monoclonal antibodies against each of the individual histones, but with different enzymatic properties. At each stage of EAE development, we see hydrolysis of H2B by a set of monoclonal antibodies in the pool of polyclonal antibodies that are able to hydrolyze H2B histone.

As for the control, it was shown even earlier that the blood of CBA mice not prone to AID does not contain abzymes hydrolyzing histones and MOG [10-13, 18-23]. Immunization of these mice with MOG leads to the production of abzymes with very weak activity that hydrolyze MOG but not histones. In addition, in mice prone to EAE up to 1-2 months of life still do not have abzymes that hydrolyze histones and MBP. Therefore, it is not possible to test your proposals using control -healthy mice.

Minor points:

Lots of acronyms without specifying them. For example, I don’t know what MOG is. Please specify all the acronyms at their first instances.

Answer: it was done

Answer: Sorry in the caption to Fig. 1B there was error, hydrolysis of H2B with mol. weight 13.8 kDa. This has been corrected in the Figure 1.

Figure 1 shows the data on histone hydrolysis and MBP with total antibodies against five histones and MBP. Hydrolysis of the histone by all antibodies results in the formation of relatively short peptides that stain weakly with Coomassie, as well as very short peptides that comes out from the gel during electrophoresis. In addition, even a large number of relatively long hydrolysis products, yield of each of which is relatively small, simply results in dirt. The effectiveness of hydrolysis can only be judged by a decrease in the amount of initial histone and MBP.

The article contains many Figures and Tables. Bringing data for all initial gels takes up a lot of space. However, taking into account your remark, we have included the full gel data in the Supplementary data – Addition to Figure 1.

In Fig6, is the data for H2A or H2B, very confusing.

Could you put Fig7-11 side by side to Fig 2-6, respectively, for easy comparison?

Answer: Sorry, but put Figures 7-11 next to Fig. 2-6 doesn't make positively sense. It is because that the number of peaks in Figures 2-6 does not coincide with the number of hydrolysis sites. Some hydrolysis sites were calculated from two peaks corresponding to these sites - the right and left peptides after hydrolysis, when both peaks are in the range of 4-14 kDa. In those cases when hydrolysis proceeds in such a way that one of the peptides after hydrolysis is less than 4 kDa, it is located in a zone that is not shown in the Figures, and the hydrolysis site is determined by MW of one peptide with a large weight. It makes no sense to give a zone of peptides less than 4 kDa, since there are a lot of peptides corresponding to double and triple peptide cleavages, which are formed after histone cleavage at only one first site. Moreover, from our point of view, if the patterns of the spectra and the pictures of hydrolysis sites are put in the order you suggested, then it will be difficult to analyze the hydrolysis sites in the Figures and tables.

Answer: Taking into account your remark, we have calculated H2B histone homology with for histones and MBP.

Text added to “Discussion”:

Therefore, it was estimated the general homology between the protein sequence of H2B with four other histones and MBP. According to five alignment, different fragments of H2B demonstrate the following comparable complete identity of amino acids between H2B and four histones H1-H4 and similarity - identical together with non-identical amino acids but with highly similar physicochemical properties (%); H1: 22.2-40.0 (45.5-72.4), H2A: 21.6-43.8 (55.7-81.2), H3: 22.1-42.9 (51.9-69.2), H4: 19.6-50 (51.7-80.0). Since IgGs against MBP efficiently hydrolyze H2B histone we estimated the homology of the MBP sequence with H2B. H2B histone has identity of amino acids with MBP 23.1-75.0 % while similarity - 46.0-87.5 %. The indicators of the amino acids identity and similarity of the sequences of H2B with all four histones and with MBP are very similar.

Thank you very much for your very helpful comments.

Best wishes

Georgy A. Nevinsky

Reviewer 2 Report

Thank you for sending me this manuscript by Andrey E. Urusov, et al to review. Overall, I judge the manuscript to be an interesting report using cross-reactivity analysis and mass spec to study the H2B hydrolysis by IgGs in EAE.

As suggestion, it would be interesting to add more information/background about auto-abs-abzymes, function, structure, etc.

In general, the way the introduction section is written gives the impression that is part of the results found in this investigation.

The manuscript should be improved because it was hard to follow the idea in some sections.

There are issues that are listed in order, as follows:

1) Page 2, line 67: change the bold “of” to normal regular style.

2) page 2, line 75: “Moreover, these changes in the differentiation profiles are bound to produce various…” here I think the word bound is not correctly used.

3) page 3, line 112: “including human herpesvirus, human endogenous retroviruses, and Epstein-Barr virus)” remove the extra bracket.

4) page 4, line 179 : “The pool of phage particles containing various antibodies on the surface was divided into ten peaks eluted from MBP-Sepharose using different…” It would be more illustrative if the authors present the figure for this result.

5) page 5, line 239: “In this work there was performed more detailed study of the catalytic cross-activity of IgGs against five individual histones and MBP in the hydrolysis of H2B histone.” It is hard to get the idea for this sentence, please rephrase the idea.

6) please include the other gels for section 2.2 purification of antibodies, aside from the supplementary Fig S4. In addition, please provide the original uncropped SDS-PAGE images.

7) some figures seem to be at low resolution and it is difficult to read some numbers (MALDI spectra figures ), provide high resolution figures

8) improve the manner to present results in table 2, table 3 and, table 4 (compacting the empty spaces)

Author Response

As suggestion, it would be interesting to add more information/background about auto-abs-abzymes, function, structure, etc.

In general, the way the introduction section is written gives the impression that is part of the results found in this investigation.

The manuscript should be improved because it was hard to follow the idea in some sections.

There are issues that are listed in order, as follows:

1) Page 2, line 67: change the bold “of” to normal regular style.

2) page 2, line 75: “Moreover, these changes in the differentiation profiles are bound to produce various…” here I think the word bound is not correctly used.

Answer: it was corrected

3) page 3, line 112: “including human herpesvirus, human endogenous retroviruses, and Epstein-Barr virus)” remove the extra bracket.

Answer: it was corrected

Answer: Sorry, but these are not the results of this article, they were published earlier with diagrams and pictures [67-73]. In this paper, they are presented to justify the fact that, as shown earlier with the use of monoclonal antibodies, their number and catalytic characteristics of Abs of patients with AIDs are very different.

Answer: it was corrected

In this work, antibodies against five individual histones for the first time were isolated from preparations of total antibodies against five histones and performed study of their catalytic cross-activity in the hydrolysis of MBP and H2B histone.

6) please include the other gels for section 2.2 purification of antibodies, aside from the supplementary Fig S4. In addition, please provide the original uncropped SDS-PAGE images.

Answer: Sorry, but there are a lot of Figures and Tables, so fig. Fig S4 has been placed in the Attached data. Original uncropped SDS-PAGE images also take up a lot of space - putting them in an article is also very problematic.

Figure 1 shows the data on histone hydrolysis and MBP with total antibodies against five histones and MBP. Hydrolysis of the histone by all antibodies results in the formation of relatively short peptides that stain weakly with Coomassie, as well as very short peptides that comes out from the gel during electrophoresis. In addition, even a large number of relatively long hydrolysis products, the yield of which is relatively small, simply results dirt. The effectiveness of hydrolysis can only be judged by a decrease in the amount of initial histone and MBP.

However, taking into account your remark, we have included the full gel data in the Supplementary data – Addition to Figure 1.

7) some figures seem to be at low resolution and it is difficult to read some numbers (MALDI spectra figures ), provide high resolution figures

Answer: All drawings are given in high resolution. However, since there are a lot of drawings, we have brought them in a small size. Considering your comment, we have increased the size of the drawings.

8) improve the manner to present results in table 2, table 3 and, table 4 (compacting the empty spaces)

Answer: It was done: Line spacing reduced to a minimum

Thank you very much for your very helpful comments.

Best wishes

Georgy A. Nevinsky