BCR-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

  1. Luqman O. Awoniyi
  2. Alexey V. Sarapulov
  3. Sara Hernández-Pérez
  4. Marika Runsala
  5. Diogo M. Cunha
  6. Blanca Tejeda-González
  7. Vid Šustar
  8. M. Özge Balci
  9. Petar Petrov
  10. Pieta K. Mattila

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Abstract

Successful B cell activation, critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodelling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through Akt and MAPK axes.

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  1. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time. The full point-by-point reply is attached together with the manuscript files.

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    Referee #3

    Evidence, reproducibility and clarity

    Review Commons recommends including the following components in referee reports:

    1. Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

    The manuscript by Awoniyi et al is an elegant study that addresses the protein composition of the lipid rafts upon BCR activation. The authors use an elegant system, employing the enzyme ascorbate peroxidase (APEX2), which in cellulo generates short-lived biotin radicals, that in turn randomly bind to proteins in their vicinity (10-20 nm) within 1 min. APEX2 is furthermore fused with the 7-amino acid sequence MGCVCSS, which allows its targeting to lipid rafts (Raft-APEX2) and with an mCherry marker. Using modern microscopy methods as well as quantitative mass-spectrometry proteomics, the authors provide a spatially and temporally resolved dynamic insight into the changes within the lipid raft and. are able to enrich multiple proteins in the lipid rafts previously not associated with BCR signaling. Furthermore, they identify Golga3 and Vti1b as proteins proximally responding to BCR activation possibly enabling vesicle transport.

    The manuscript is generally well written, the study is well-conceived and well-controlled. Nevertheless, the authors may answer some important questions (see below)

    Major comments:

    • Are the key conclusions convincing?

    Yes, the key conclusions of the study are convincing and based on elegant experiments

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    • In Figure 2, the HEL-specific A20 B cells are stimulated with anti-IgM F(ab)'2. While, beyond a doubt, anti-IgM F(ab)'2 is a potent stimulus, which triggers BCR signaling activation, I am curious why the authors chose it over the HEL antigen.

    • Describing figures 4 and 5, the authors state that they did not identify prominent BCR signaling pathway regulators. My major concern here is that the authors employ cancerous B cells for their analyses. The lipid raft composition and proteins recruited to the rafts in these cells may vary from those in primary wild-type B cells. While the authors do keep in mind that the signaling protein composition may vary between cell lines, it may vary even more between lymphoma B cell line and primary wild-type cells. Therefore, it may be beneficial to verify the expression of the "unexpected" proteins, such as Golga3, Kif20a, and Vtib1b in primary cells using immunofluorescence analyses similar to the ones presented in Fig 6.

    • The authors mention in the discussion, that Syk was not identified in their data set. This is surprising as Syk has been attributed with an important role in the proximal BCR signaling (Kulathu et al, https://doi.org/10.1111/j.1600-065X.2009.00837.x)

    • Would it be possible to detect Syk using the immunofluorescence technique from Figure 6?

    • Additionally, as stated in the text, A20 cells express endogenous IgG2a. Have the authors tried to conduct similar experiments stimulating with anti-IgG antibodies instead of anti-IgM F(ab)'2?

    • Have the authors tried to co-express the IgD-BCR to mimic mature peripheral B cells?

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    I cannot estimate the cost but if conducted, some experiments might take several months

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes

    Minor comments:

    • Specific experimental issues that are easily addressable.

    • Are prior studies referenced appropriately?

    Yes

    • Are the text and figures clear and accurate?

    Yes, but, if possible, please provide the data instead of writing "data not shown"

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Fig. 1D and Supp. Fig. S1C: the authors state that after IgM cross-linking, the non-transfected and Raft-APEX2-transfected cells showed "indistinguishable" p-Tyr levels. From my perspective, the Raft-APEX2-transfected cells show higher levels of p-Tyr. It is possible to quantify it?

    Some paragraph titles are very short and descriptive (e.g. Proteomic analysis, membrane-proximal proteome etc.). It could improve the reading if the paragraph titles consisted of respective key findings

    Significance

    2. Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    • This study dissects in a spatio-temporal manner the early events upon BCR stimulation and the enrichment of various proteins in the vicinity of lipid rafts. While conceptually not novel, the study provides novel methodology to address this question. This is technically relevant and worth to be published after a major revision.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    *This study prominently overlooks a bulk of literature that supports the BCR dissociation activation model and does not comment that (reviewed in Maity et al, Volume 1853, Issue 4, April 2015, Pages 830-840)

    • State what audience might be interested in and influenced by the reported findings.

    The findings of this manuscript are specifically interesting for researchers who study early events of B cells activation, specifically the changes in the membrane composition and early BCR signaling

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Immunology, Molecular and Cell Biology

    Referee Cross-commenting

    I agree totally that "the article would greatly benefit from a follow-up investigation on the functional/physiological relevance of the proposed players", however only if this is easily done with the CRISP mediated knock out as mentioned by both reviewers. In addition it s interesting to see data on cells stimulated with the antigen instead of anti IgM fab'2.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Awoniyi et al. utilizes APEX2-mediated proximity proteomics to investigate the protein composition of lipid rafts and their dynamics in the context of B cell receptor (BCR) signaling. The authors add a 7 amino acid guide sequence to an APEX2-mCherry construct to specifically target the fusion protein into lipid raft plasma membrane domains and thereby spatially label and identify contained proteins. While a larger number of lipid raft-related proteins were verified, the study focuses on a smaller subset that is proposed to be specifically related to BCR signaling. Unexpectedly, this approach suggests key players of BCR signaling to be excluded from lipid rafts in an inducible manner. Finally, two of the identified proteins, Golga3 and Vti1b, were further investigated by immunofluorescence microscopy. Since both proteins are shown to be recruited to the plasma membrane and to colocalize with antigen, the authors propose Golga3 and Vti1b as novel targets of BCR activation and drivers of subsequent antigen internalization.

    Major Comments

    • A major claim of this study is that the majority of BCR signaling proteins (including CD79a and CD79b as parts of the BCR as well as BLNK) get excluded from lipid rafts upon stimulation. Moreover, many components of the endocytosis/vesicle trafficking pathways have been identified and the authors raise interesting points regarding the BCR as signaling platform versus the BCR as antigen internalization complex. This is intriguing and could even be explored further (e.g. by presenting Figure S3 in the main manuscript). However, the claim that Vti1b and Golga3 (and possibly Kif20) play key roles in the endocytic processes underlying BCR/antigen endocytosis and subsequent processing needs further verification e.g. by gene targeting experiments. In its present form, the manuscript links these proteins to B cell activation but does not convincingly back up the implied functional relevance to antigen/BCR endocytosis and/or trafficking leading to antigen presentation via MHC II.
    • It should be explained why the proteomic experiments were conducted using anti-IgM antibodies as opposed to the more physiological stimulation via HEL antigen, used for the microscopy studies.
    • Even though it is the central approach, the number of figures derived from the APEX2 proteomic experiments is quite high and should be condensed. For example, Figures 4 and 5 could be merged.
    • Figure 1D/E and Figure S1C seem to be the same pictures.
    • In contrast to Golga3, Vti1b is not mentioned in Figure 4 and the authors should explain why this particular protein was chosen for further investigation among all others (as opposed to proteins enriched upon anti-IgM treatment).
    • In Figures 6 and S4, the most apparent changes in Golga3 staining appear to be the increased (cytoplasmic and peripheral) vesicle size and intensity. For the analysis and quantitation of peripheral Golga3 staining, a tubulin-based masking algorithm was used to segment the image. This raises three concerns: 1) The tubulin staining that was used for masking appears to be rather blurry and the expected microtubule network is barely visible. 2) More information is needed on how the masking algorithm treated Golga3 vesicles touching the mask border. Based on the images in S4, there seems to be substantial overlap between (visibly peripheral) Golga3 vesicles and tubulin, so this will likely have an impact on quantification results. 3) Authors should comment on the overall increase in Golga3 upon activation.

    Minor Comments

    • While the APEX2 construct is globally targeted into the lipid raft environment, the study uses this approach to investigate proteins that are in the proximity of the IgM-BCR. The authors mention in the discussion that there have been "challenges" to target the BCR directly. It may be beneficial to briefly discuss those problems the authors have been facing with.
    • Figure 5B: It will be informative to show the BCR-induced (fold change) enrichment of Golga3 and Vti1b (and Kif20) in relation to IgM /kappa LC and the "classical" BCR signaling-related players.
    • Figure 6AB: Please clearly indicate that unmasked images are displayed, but masked images were quantified for Golga3 staining.
    • Figure 6CD: Since the quantification protocol of vesicle positioning involves nuclear staining, please depict respective DAPI stainings.
    • Figure S1D: It should be indicated that AF633-streptavidin was used for the flow cytometry experiment in Figure S1D (x axis).

    Significance

    Overall, the presented study offers an interesting approach and provides a novel, unbiased view on BCR-mediated lipid raft dynamics. The method is appealing in its technicality and its presentation, and hence, might attract the attention of a larger community working on plasma membrane localization of signaling platforms. It proposes two candidate signaling proteins and verifies their BCR-dependent colocalization with lipid rafts and antigen. While Golga3 and Vti1b are novel and interesting target proteins in the context of BCR activation, a functional assessment of these proteins is not presented. Certainly, the article would greatly benefit from a follow-up investigation on the functional/physiological relevance of the proposed players. As it stands, the manuscript largely remains on the level of exploration.

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    Referee #1

    Evidence, reproducibility and clarity

    This is an interesting study identifying membrane RAFT associated proteins in a mouse B cell line, before and after BCR stimulation, using a proximity biotinylation method. This method relies on the expression of an biotinylating enzyme APEX2 with a RAFT targeting domain that is activated by H2O2 addition. The system is well controlled by comparing transfected to non-transfected cells and by titrating H2O2 etc. The expected anti-IgM induced recruitment of the BCR to the RAFT domain is well documented in this system. The authors identify by proteome analysis over 1600 proteins in proximity to the membrane-targeted APEX2, most of which are constitutively labelled, i.e. do not change upon BCR stimulation. Only a minority of proteins (less than 100) changes dynamically between resting and stimulated state. Some results are surprising, as the authors discuss, as the known players of the BCR signalling pathway hardly change in their Raft association. Interesting is also the exclusion of signalling proteins such as Btk, BLNK and Ig-alpha/Ig-beta from BCR clusters upon activation. The strength of the system is that the APEX2 system causes a biotinylation with 1 min, which is an advantage to other systems and that the authors analyse 3 time points after BCR stimulation. The data are thoroughly analysed and discussed. The following points should be addressed:

    1. Why did the authors not use the HEL antigen to stimulate their cells, as the A20 line expresses a Ag-specific BCR? Would this not be more physiological than Fab2-anti IgM?
    2. Many proteins of pathways like RNA transport, Spliceosome, mRNA surveillance, mismatch repair etc. are identified. Although the authors try to explain some of these data, they should also consider unspecific labelling or unspecific enrichment of these proteins which have nothing to do with raft association. This should be more openly discussed.
    3. The authors follow up two proteins that dynamically change during activation, Golga3 and Vti1b, and demonstrate their membrane association upon activation. This is of course relevant. What is missing, is some genetic studies. CRISPR-mediated KO is not difficult to do in cell lines. Have the authors produced such mutants for these two genes and analysed possible phenotypes in BCR signalling or other aspects? This would certainly strengthen the study.

    Significance

    This is a unique approach to globally identify proteins associated with the BCR in Rafts upon BCR stimulation. Comparable studies with other methods have been published before for B cell lines. Gupta et al. used quantitative proteomics of isolated RAFT-associated proteins before and after BCR stimulation. They also found that the association of most proteins to RAFTs was not changed after BCR stimulation. Saeki et al. used a proteomic approach in another B cell line to identify RAFT associated proteins, but without comparing stimulated to unstimulated cells. The approach used here has the advantage of not selecting only membrane bound proteins, but equally identifiying cytosolic proteins in vicinity to the Raft as well. Furthermore, dynamic changes are better analysed than in the other two studies. Therefore, the findings are relevant and a good advance in the field.

  5. Version published to 10.1101/2020.09.29.318766v4 on bioRxiv
  6. Version published to 10.1101/2020.09.29.318766v3 on bioRxiv
  7. Version published to 10.1101/2020.09.29.318766v2 on bioRxiv
  8. Version published to 10.1101/2020.09.29.318766v1 on bioRxiv