NMR Studies of the Interactions between Sialyllactoses and the Polysialytransferase Domain for Polysialylation Inhibition

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The objective of this study is to investigate the impact of SL concentration on NCAM polysialylation by analyzing the interaction between PSTD and 3’-SL/6’-SL. The authors utilized NMR spectroscopy to suggest theoretical concentrations for infant formula supplements. Additionally, the study proposes that SLs have a dual effect on the interaction with PSTD, serving as a beneficial food source for infant formula and gut health while potentially preventing the overexpression of polysialic acids that can lead to cancer cell migration.

The authors apply a modern experimental technique, the discussion and results are in mutual agreement.

 

Minor Recommendations:

The authors could provide the structure of N-Acetylneuraminic acid, the most common of the sialic acids, as well as the structures of 3'-N-acetylneuraminyl-D-lactose and 6'-N-acetylneuraminyl-D-lactose in the introductory part.

The authors could present the part from the introduction (line 45 - line 63) schematically, which would make it easier to follow the presented text.

In a few sentences they should explain the HSQC NMR method and state the advantage of this method.

On Figures 2 and 5, mark significant differences between a and b parts (in b part V251, V273, 1275, K248).

In Figures 3, 6 and 7, mark the error in determining the chemical shift.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript „The NMR Studies of the Interactions between Sialyllactoses and Polysialytransferase Domain for the Polysialylation Inhibition” is a next work in a series of reports on Polysialytransferase Domain.

The idea of using NMR shift changes to study molecular interactions is very interesting. However, in several places in the manuscript, the inhibitory activity of compounds is related to such results. Such approach needs more explanation on the NMR studies (competition?) and relation to data obtained from other methods. For example, describing heparin, the Authors refer to [29] with a phrase “heparin LWMH has been found to be an efficient inhibitor due to its stronger binding to the PSTD”, whereas the results were based on spectroscopic determination of interactions, not functional experiments (from [29]: The findings in the present study demonstrate that PSTD domain is a potential target of heparin). In line 165, please specify the inhibitory effect and how it was established using NMR. How do the CSPs translate to the strength of inhibitory effect (line 175)? The caption to Fig. 7 indicates comparison of the inhibition, whereas the data show structural interaction consequences?

In describing the purpose of the research (lines 70-73) the Authors mention “In this study, our interest is to understand how SL's concentration level effects on NCAM polysialylation through the modulation of the interaction between the PSTD and 3’-SL/6’-SL, and proposed their theoretical concentrations for the supplements of infant formula using NMR spectroscopy”. Then, the presented research describes the interactions (NMR studies), no experiments on actual NCAM polysialylation were planned or performed. Either the aim of the study should be adjusted to the planned experiments, or other data (or explanation) provided.

The next sentence (lines 73-76) is quite attractive, but does not find support in the following data. It could be misinterpreted by the readers of the paper.

The sentence (line 242) “In practical application, it’s enough to take above 1 mM 6’-SL for the inhibition of the PSTD-(CMP-Sia) interaction” is not confirmed by actual inhibition, but similarity of structural data (structural or functional consequences), please reconsider such statement.

In general, the NMR experiments were performed for specific pairs (PSTD and another substance). Therefore an explanation for selection of data for specific panels would be interesting (0.5, 1,2,3 mM). No data on three-component mixtures are provided, did the Authors consider such possibility?

The interaction with helical structure could be related to orientation of amino acids (helical wheel) and their chemical character. In the PSTD, the number of basic residues is high, does this have any possible effect on the interactions?

The concentration of 3’-SL in human milk during lactation is directly compared to the concentrations used in the experiment. It would be true for considering the sialylation in milk, but not in the cells (digestion, transfer etc. losses). Please comment on the concentration effects and digestion. The sentence (line 388) is strange: This is why the maximum concentration of 3’-SL and 6’-SL in human milk during lactation are naturally chosen to be 220 mg/L (close to 0.5 mM) and 500 mg/L (about 1.0 mM), respectively. Please clarify this statement.

 

There are several problems in the Materials and Methods:

Line 336: The PSTD (246K-277R) should be a 32 amino acid sequence peptide

Line 341: In which, the PSTD sequence is labelled by underline.

Peptide PSTD contains several basic residues. How was the concentration established for CD and NMR studies? Which counterions were in the sample and how their weight was calculated?

Line 351: 25% tetrafluoroethylene (TFE) – please check which “TFE” compound was used for CD experiment.

Line 351: Please confirm 8.0 uM concentration (ultramolar?)

Line 361: D2O (index). The buffer was prepared using regular water of deuterated water?

Line 354: biomolecule-protein (DNA) (?)

The formula in 380 needs better representtaion, to differentiate indices and mathematical operations. The shifts in 2D experiment were separated between H values and N values? Please clarify.

 

Minor issues:

 

In the Abstract, the amount of introductory material is overwhelming the results of this research paper.

The reference for VEGF results should be included around line 40, as it is not certain that ref. 10 includes all this information.

The quantitative information on SL content in human milk would be interesting in Introduction (lines 65-67), not only in the Discussion.

The sentence (line 60) is not clear “More recently, cytidine monophosphate (CMP) was found that polysialylation could be partially inhibited when CMP-Sia and polySia co-exist in solution”

Line 79: please rephrase: that the alpha-helical content contains 26.5% of the PSTD in the absence of any ligands

The word “significant” is used several times in the text, please provide the information on statistical significance of the results or modify the text.

Figure 1. In PDF, the blue line looks black. The legend (compound abbreviations) does not correspond to the caption. Please confirm the concentration (uM) both in the caption and in Methods.

Table 1. The current format of the table may be modified to provide a common line with that the helical content of the PSTD, as now it is not easy to read.

Line 57: “The PSTD is 32 amino acids unique to the .2,8-polysialyltransferases (polyST),” – this describes a peptide, not just amino acids, please rephrase the sentence and check the text for similar phrases.

In NMR figures, the values at F2 (ppm) have practically invisible decimal point (.), which may be confusing. For overlaid spectra, the color change should be explained, either as the result of overlay, or spectral specifics.

In CSP figures, the numbers correspond to amino acid reside numbers in PSTD sequence, sequence would require the use of amino acid symbols. The color scheme should be re-considered, to keep the same data in the same color (figure 3, 0.5 mM 3’-SL,).

Position of Table 3 may be confusing.

 

 

Other comments:

There are several places when plural nouns are combined with verbs in singular form: line 12: Sialylated milk oligosaccharides (SMOs) is the most important oligosaccharides.

Line 169: LNWH

Line 238 and other parts of the text: “the CSPs for the PSTD-(1 mM 6’-SL) interaction are less than that for the PSTD- 1mM CMP interaction”: are CSPs countable or not (less or smaller?)

Line 260: Because CMP-Sia couldn’t be bound to the long helix H2. This sentence seems unfinished.

Line 339: in peptide chemistry, termini are used instead of terminals.

Comments on the Quality of English Language

The comments are included in the above list

Author Response

Please see the attachment.

Author Response File: Author Response.pdf