EMBO reports - Peer Review Process File - EMBO-2017-44351
Manuscript EMBO-2017-44351
Regulation of perforin activation and pre-synaptic toxicity through C-terminal glycosylation Imran G. House, Colin M. House, Amelia J. Brennan, Omer Gilan, Mark A. Dawson, James C. Whisstock, Ruby H.P. Law, Joseph A. Trapani, and Ilia Voskoboinik Corresponding author: Ilia Voskoboinik, Peter MacCallum Cancer Centre
Review timeline:
Submission date: Editorial Decision: Revision received: Editorial Decision: Revision received: Accepted:
11 April 2017 02 May 2017 24 May 2017 23 June 2017 25 June 2017 27 June 2017
Editor: Achim Breiling Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)
1st Editorial Decision
02 May 2017
Thank you for the submission of your research manuscript to EMBO reports. We have now received the reports from the three referees that were asked to evaluate your study, which can be found at the end of this email. As you will see, all three referees highlight the potential interest of the findings. However, referees #2 and #3 have raised a number of concerns and suggestions to improve the manuscript, or to strengthen the data and the conclusions drawn. As the reports are below, I will not detail them here. Given the constructive referee comments, we would like to invite you to revise your manuscript with the understanding that all referee concerns must be addressed in the revised manuscript and in a point-by-point response. Acceptance of your manuscript will depend on a positive outcome of a second round of review. It is EMBO reports policy to allow a single round of revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript. Revised manuscripts should be submitted within three months of a request for revision; they will otherwise be treated as new submissions. Please contact us if a 3-months time frame is not sufficient for the revisions so that we can discuss the revisions further. Supplementary/additional data: The Expanded View format, which will be displayed in the main HTML of the paper in a collapsible format, has replaced the Supplementary information. You can submit up to 5 images as Expanded View. Please follow the nomenclature Figure EV1, Figure EV2
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etc. The figure legend for these should be included in the main manuscript document file in a section called Expanded View Figure Legends after the main Figure Legends section. Additional Supplementary material should be supplied as a single pdf labeled Appendix. The Appendix includes a table of content on the first page, all figures and their legends. Please follow the nomenclature Appendix Figure Sx throughout the text and also label the figures according to this nomenclature. For more details please refer to our guide to authors. Important: All materials and methods should be included in the main manuscript file. Regarding data quantification and statistics, can you please specify, where applicable, the number "n" for how many experiments were performed, the bars and error bars (e.g. SEM, SD) and the test used to calculate p-values in the respective figure legends. Please provide statistical testing where applicable. We now strongly encourage the publication of original source data with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure or per figure panel. When submitting your revised manuscript, we will require: - a complete author checklist, which you can download from our author guidelines (http://embor.embopress.org/authorguide#revision). Please insert page numbers in the checklist to indicate where the requested information can be found. - a letter detailing your responses to the referee comments in Word format (.doc) - a Microsoft Word file (.doc) of the revised manuscript text - editable TIFF or EPS-formatted single figure files in high resolution (for main figures and EV figures) I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. ---------------------------REFEREE REPORTS ---------------------------Referee #1: In "Regulation of perforin activation and pre-synaptic toxicity through C-terminal glycosylation", House et al. present evidence that glycosylation of the C-terminal extension of full-length perforin prevents calcium dependent oligomerization of the protein at neutral pH. This would presumably protect cytotoxic lymphocytes from their own perforin as it transits through the ER. The authors' data also provide insight into how the autoinhibitory extension of perforin is removed after transit into low pH lytic granules, suggesting that a diverse set of lysosomal proteases cooperate to do the job. Taken together with the Voskoboinik lab's previous paper on this topic (Immunity 34, 879-892), this work puts forth an elegant model explaining how perforin is generated without harming its parent cell. Overall, I found the experiments presented here to be insightful and supportive of this proposed model. Data were well-presented and appropriately interpreted, and the Discussion was a pleasure to read. I must say I have little to add other than to commend the authors on some nice biochemistry. ---------------------------Referee #2: The author present a convincing case that perforin is produced as a proform that requires C-terminal proteolytic processing to remove a C-termnal glycopeptide prior to acquiring pore forming activity
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(urea recoverable) at neutral pH in the presence of Calcium. The work relies on mass spec for acurate mass of material from cell lines and primary cells. They have perforin deficient cell lines for expression and testing of engineered perforin. They found that there was no specific proteolytic site, but a preferred site probably due to flexibility of the peptide. Removal of the glycan my mutation or proteolysis didn't alter membrane binding, but was required for oligomerization. There is a minor issue with the discussion of insect cell derived material, which has full activity without removal of the glycol. A little more discussion of the yeast glycans could be helpful. The ER high mannose oligosaccharide is similar in yeast and mammals so protection in the ER is probably the same, but the processing in the Golgi apparatus is different and generally results in triimming down to 3 mannose residues in the Golgi. There is calcium in the Golgi, although it has a lower pH than the ER, which may save the insect cells from the perforin. The authors could state with some confidence that the 5-9 mannose structures in endo H sensitive glycans must be sufficient to inhibit oligomerisation, whereas the 3 mannose structures in insect cells don't prevent oligomerisation. See: PMID 17979671 ---------------------------Referee #3: The work by House et al. shows some well-executed assays on the molecular characteristics of perforin, leading to self-cell protection during its expression and targeting to the lytic granules. The manuscript is easily readable and well-written and the methods described are clear. Some of these results complement their previous work on perforin sorting and maybe of relevance for the fields of immunology and cell biology. Other issues seems to be already addressed elsewhere (reference 19), such as those depicted for mutant Asn549, which was called Gly2 mutant in the previous report. The C-terminal mutants showed in the previous report provoked autolysis and different problems in their sorting from the ER and TGN. They also showed differential resistance to EndoH and PNGaseF treatment. In general, the information from Mass Spectrometry experiments is interesting, but it is not well developed and there is not enough data on cell and protein functionality to support the claims stated by the authors. The oligomerization assays do not sustain the conclusions raised. The Asn549 mutant has not been analyzed by MS, which difficult the comparison with WT and the other mutants for prPRF and flPRF presence, basic for some studies. Western blot is used for comparison of little amounts of proteins instead, but with no quantification at all. Major comments: 1. To sustain statements from Figure 1, it would be really useful to see images (immunofluorescence) correlating the localization of WT and mutant perforins and some subcellular markers in cells KHYG1 KO expressing these proteins at different times of sorting or pulse-chase experiments to assess the localization of the different mutants. Same applies for Asn54. 2. From data shown in Figure 2, it seems that mutants in 2D, 2E and 2F are processed differently from WT. The amount of protein detected at different peaks (Met541, Leu543; as in the wt protein or Ser-mutated) is really similar to the Leu542 specie from WT. Moreover, the one in E even accumulates flPRF. This is somewhat striking and may indicate a requirement for correct cleavage (even if the final result is functional and non-toxic, what we do not really know). Authors may show the extent of cell death and autolysis for cells expressing each of the mutants and wt perforin. Please, comment in the text. 3. Figure 3 do not correspond to data generated in this work; can be included as a part of other figure (2), as a supplemental or even excluded, since it does not have entity for being a complete Figure. 4. EndoH and PNGase F activities might be synergistic (Figure 5A). Can authors probe whether their effects are additive? Indeed, it seems that electrophoretic mobility of PNGase F-treated flPRF is higher than prPRF (Figure 5A, WB DMSO and CNCA). Therefore, the electrophoresis mobility of the Asn549Gln flPRF mutant, not able to be N-glycosylated, should be higher than prPRF, but it
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is clear that residue 205 is also affecting. From the experimentation showed in reference 19 by the same authors, the 205+549 mutation prevented both, N-glycosylation and retained mobility in electrophoresis (Gly1+Gly2), but additionally, affected to the cell lysis activity of cells expressing it (although not to the in vitro activity of recombinant perforin protein). Those effects could be reversed by a different new N-glycosylation site by mutagenesis of this mutant. Therefore, it is striking the statement made by the authors about the ability of the 549 mutant in cell killing. The mutant Asn549 seems to have a little decrease of activity, since the % of specific lysis showed at Figure 5B demonstrates the need of higher amounts of protein for same extent of lysis, as was observed in the experimentation with Gly2 mutant in the reference 19. These experiments shall be performed in parallel with WT. It seems that the E64D effect on WT is abolished upon 549 mutation. 5. The oligomerization assay is not really accurately demonstrating that there is no oligomerization, since the Asn549 mutant behavior basically phenocopies the one of the WT. It seems, at least, as efficient as the WT in the recovering of prPRF with Urea in Ca2+ presence (therefore in oligomerization). In fact, it is surprising that authors could detect flPRF in Figure 5F, since they state that it is not present in Figure 5B. Indeed, why is then so toxic? How is it activated? Minor points: Can authors show the PCRs for KHYG1 CRIsP/Cas perforin WT/KO clones? Please, clearly indicate the strategy and sequences for gene editing. In page 5, authors describe several 'shorter perforin molecules' than the one that terminates at Leu 542 as ending at Leu 543 and Gly 544. These seem to be too long to be shorter. Please, clarify.
1st Revision - authors' response
24 May 2017
REVIEWER #1 Thank you for your kind comments. REVIEWER #2 There is a minor issue with the discussion of insect cell derived material, which has full activity without removal of the glycan. A little more discussion of the yeast glycans could be helpful. The ER high mannose oligosaccharide is similar in yeast and mammals so protection in the ER is probably the same, but the processing in the Golgi apparatus is different and generally results in triimming down to 3 mannose residues in the Golgi. There is calcium in the Golgi, although it has a lower pH than the ER, which may save the insect cells from the perforin. The authors could state with some confidence that the 5-9 mannose structures in endo H sensitive glycans must be sufficient to inhibit oligomerisation, whereas the 3 mannose structures in insect cells don't prevent oligomerisation. See: PMID 17979671 We assume that the Reviewer is referring to “insect”, not “yeast”, glycosylation (we never used yeast to purify perforin). Otherwise, we absolutely agree with this comment, and have made appropriate changes in the text (page 11). REVIEWER #3 In general, the information from Mass Spectrometry experiments is interesting, but it is not well developed and there is not enough data on cell and protein functionality to support the claims stated by the authors. The oligomerization assays do not sustain the conclusions raised. The Asn549 mutant has not been analyzed by MS, which difficult the comparison with WT and the other mutants for prPRF and flPRF presence, basic for some studies.
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Western blot is used for comparison of little amounts of proteins instead, but with no quantification at all. With respect, we disagree with these very broad and negative comments, which are clearly at odds with expert Reviewers 1 and 2, who had no such issues. We feel that to argue that the MS is not well developed is very unfair – this is the first study since perforin was discovered in the mid- 1980s where different forms of endogenous perforin were precisely distinguished and identified. The results were reproducible, with very similar findings in a natural killer cell line and in primary human natural killer cells. It is possible though that the Reviewer has overlooked some of the data, and we kindly refer them to Figures 1A,B, Figure 2 and Appendix Figure 1. Major comments: 1.
To sustain statements from Figure 1, it would be really useful to see images (immunofluorescence) correlating the localization of WT and mutant perforins and some subcellular markers in cells KHYG1 KO expressing these proteins at different times of sorting or pulse-chase experiments to assess the localization of the different mutants. Same applies for Asn549.
It is unclear to us from the either the Reviewer’s comment or from a purely scientific standpoint why it would be important to conduct the suggested experiments on over a dozen mutants (which will take many months to complete), for the purpose of the current study. To be clear: this manuscript is strictly about perforin processing, not about perforin trafficking or expression. We have provided unequivocal physical evidence using MS and Western analysis that the mutants are processed (Figure 2). We would also like to refer the reviewer to Figures 4A/B and 4D/E, which offer a convincing negative control for impaired perforin processing by inhibiting H+-ATPases (with concanamycin A) or cysteine proteases (with E64D). The functional consequences of such inhibition are shown in Figures 2C, 2F, respectively. Asn549Gln has been assessed exhaustively using IF microscopy, Western analysis and a variety of functional assays in Brennan et al (Immunity 2011, Figs 5 and S5). Below, as requested we have supplied a deconvolution of an MS spectrum of perforin from Asn549Gln-expressing cells, which confirms that its processing is essentially indistinguishable from wild-type perforin, and this is absolutely consistent with that previously published (Brennan et al, 2011) and the current data.
L542
W555 Q540
It is unequivocal that the cleavage of perforin can only occur in the granules, based on Figures 1A/B (and related Figure EV4), 4A/B (and related Appendix Figure 1). The only instance where perforin cleavage appears to be impaired is in the case of severely misfolded mutants, eg see Chia et al (PNAS 2009, Blood 2012) or Risma et al (J Clin Invest, 2006) or under temperature-restricted conditions (Brennan et al., 2011).
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2.
From data shown in Figure 2, it seems that mutants in 2D, 2E and 2F are processed differently from WT. The amount of protein detected at different peaks (Met541, Leu543; as in the wt protein or Ser-mutated) is really similar to the Leu542 specie from WT. Moreover, the one in E even accumulates flPRF. This is somewhat striking and may indicate a requirement for correct cleavage (even if the final result is functional and non-toxic, what we do not really know). Authors may show the extent of cell death and autolysis for cells expressing each of the mutants and wt perforin. Please, comment in the text.
The Reviewer is correct in that there are some slight differences between these mutants and the wild-type perforin. This is not surprising as we mutate residues around Leu542/543, where most of the processed perforin molecules normally terminate. However, the key point of these experiments was to demonstrate the lack of a unique consensus cleavage site, ie none of the many mutations we introduced precludes the removal of the C-terminal glycan. Furthermore, despite the mutations, perforin was never trimmed any further back from the C terminus than Q540 – this is consistent with the X-ray crystal structure shown in Figure 3. Our results in Figure 2A-F clearly demonstrate that despite the mutations, the C-terminal tail (and attached glycan) is being removed, and the dominant C-terminal residues were either Leu(Ser)542 (Figures 2A-C) or Met541 (Figure 2D) or Leu(Ser)543 (Figures 2E, F). Please note that some confusion might have occurred, as each plot shown in Figure 2 also includes untreated control (shown as a solid trace). We have now clarified this in the text. With respect to a potential accumulation of some amount of full-length perforin in Figure 2E, there are two potential explanations: (i) this may be a sample-sample variation (eg see Figure EV2 or replicates 1 and 4 in Appendix Figure 1 where some full-length perforin is still present), or (ii) given the importance of the C-terminus for perforin export from the ER and considering the fact that we used whole cell lysate for MS, it is conceivable that there may be some minor retention of fulllength perforin in the ER, but this will be all but impossible to validate. Multiple Western blots of the type shown in Figure 1E have not indicated that the mutant shown in Figure 2E accumulates more full-length perforin. Finally, we cannot demonstrate the extent of cell death or autolysis, as these cells were stably virally transduced and selected in culture - autolysis can only be estimated when de novo synthesis occurs eg with transient transfection (see Brennan et al, 2011). It is unclear to us why we should repeat experiments where the data was originally published by us (Brennan et al., Immunity 2011), and when the issue of perforin trafficking has been resolved. Once again, irrespective of the mutations introduced into the C-terminus, perforin is still processed (please compare with CNCA or E64D treated cells as shown in Figures 4B/E). 3.
Figure 3 do not correspond to data generated in this work; can be included as a part of other figure (2), as a supplemental or even excluded, since it does not have entity for being a complete Figure.
This is an important figure as it substantiates and illustrates the structural basis of our experimental observations, and we feel that having this Figure as a part of the main text will be valuable for understanding the paper. Furthermore, we have never published/emphasised the structure of the carboxy-terminal peptide. As this Figure does not fit the format of Figure 2, we have reduced its dimensions to make it less prominent, and we hope that the Reviewer will not have strong objections against this. 4. For clarity, we split Question 4 into three parts. 5. EndoH and PNGase F activities might be synergistic (Figure 5A). Can authors probe whether their effects are additive? This is impossible. PNGaseF is a peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase and, therefore, cleaves between the innermost GlcNAc of N-linked glycans and asparagine thus removing the glycan totally. In contrast, EndoH cannot process complex glycans and, only removes N-linked mannose-rich oligosaccharides. Our results shown in Figure 5A clearly demonstrate that EndoH does not activate flPRF to the same extent as PNGaseF, consistent with the substrate specificity of the two enzymes and in agreement with the presence of two forms of perforin, which are selectively sensitive or insensitive to EndoH (see Western immunoblot in Figure 5A).
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Indeed, it seems that electrophoretic mobility of PNGase F-treated flPRF is higher than prPRF (Figure 5A, WB DMSO and CNCA). I hope we have understood this comment correctly. It should be clear that since CNCA-treated perforin is longer than DMSO-treated perforin, which has the last 12-15aa proteolytically removed, the former has a slower mobility than the latter. In fact, PNGase-treated “prPRF” has two bands – one from the cleaved (lower band) and one from the small amount of full-length (upper band) perforin. The upper band there appears to be the same as the CNCA-treated deglycosylated (PNGaseF) perforin. Therefore, the electrophoresis mobility of the Asn549Gln flPRF mutant, not able to be Nglycosylated, should be higher than prPRF, but it is clear that residue 205 is also affecting. From the experimentation showed in reference 19 by the same authors, the 205+549 mutation prevented both, N-glycosylation and retained mobility in electrophoresis (Gly1+Gly2), but additionally, affected to the cell lysis activity of cells expressing it (although not to the in vitro activity of recombinant perforin protein). Those effects could be reversed by a different new N-glycosylation site by mutagenesis of this mutant. Therefore, it is striking the statement made by the authors about the ability of the 549 mutant in cell killing. We apologise in advance if we misunderstood this comment, but in the experiments shown in Ref 19, perforin-transfected cells were selected for identical protein expression using fluorescent reporter (please see Materials and Methods in that paper). Therefore, we had accounted for the death of host lymphocytes. As shown in Ref 19 (Fig. 2C, D), Gly2 (Asn549Gln) mutant has wild-type cytotoxic activity and trafficking, but it is also toxic to the host cells as compared to wild-type or non-functional perforin (see Fig 2A, S2 in Ref 19) due to its constitutive cytotoxic (as shown in the current study), when in the calcium-rich ER. The mutant Asn549 seems to have a little decrease of activity, since the % of specific lysis showed at Figure 5B demonstrates the need of higher amounts of protein for same extent of lysis, as was observed in the experimentation with Gly2 mutant in the reference 19. These experiments shall be performed in parallel with WT. It seems that the E64D effect on WT is abolished upon 549 mutation. With respect, it is impossible to compare the results in the current paper, where we assessed enriched native perforin, with those in Ref 19, where we used purified recombinant perforin (using baculovirus; Fig 1 in Ref 19). Also, as shown in that reference, using a variety of cell-free and cellbased experimental systems, Asn549 has equal cytotoxic function to the wild-type protein. We feel that the data shown in Figures 4C, 4F, 5 and Appendix Figure 2 convincingly demonstrate the inhibitory effect of the C-terminal glycosylation on perforin function. In any case, we struggle to understand, how those suggested parallel experiments would influence the conclusions of the current study. The Reviewer is right – E64D does not inhibit Asn549 activity, since that mutant has no C-terminal glycan. This is precisely what we have shown and discussed in the current paper. 6.
The oligomerization assay is not really accurately demonstrating that there is no oligomerization, since the Asn549 mutant behavior basically phenocopies the one of the WT. It seems, at least, as efficient as the WT in the recovering of prPRF with Urea in Ca2+ presence (therefore in oligomerization). In fact, it is surprising that authors could detect flPRF in Figure 5F, since they state that it is not present in Figure 5B. Indeed, why is then so toxic? How is it activated?
We very respectfully disagree with the Reviewer and suggest that perhaps the data in question has been misunderstood. A further clarification is therefore here given. As can be seen in Figure 5B and below, incubation of isolated WT perforin with target cells leads to the loss of the perforin band representing proteolytically processed perforin (prPRF) that lacks the inhibitory C-terminal glycan. This loss of signal is due to generation of SDS-resistant oligomers (please see Baran et al, Immunity 2009). However, this is not seen for flPRF (shown below). It is clear that the difference between the levels of prPRF at 0’ incubation and 20’ is quite significant.
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Upon addition of chaotropic urea to the protein lysate, perforin oligomers are disrupted and prPRF is recovered, as can be seen in the remainder of Figure 5B.
In stark contrast, when the experiment is repeated with Asn549Gln perforin (lacking the C-terminal glycan), both flPRF and prPRF (indicated with arrows below) form SDS-resistant pores, demonstrated by a loss of both bands on Western blot.
To substantiate this further, please, see our data below, where we show, once again, an intact high molecular weight band in DMSO, CNCA and also in E64D treated cells, and the disappearance of that signal in the case of the Asn549 mutant. Once again, this is consistent with our functional data shown in Figures 4, 5 and Appendix Figure 2. 8M Urea - Ca2+
+ Ca2+
- Ca2+
0’ 20’
0’ 20’
0’
+ Ca2+
20’ 0’ 20’
DMSO
CNCA
E64D
Asn549
The reviewer is correct in saying that oligomerised WT and Asn549Gln perforins are essentially phenocopies following urea treatment – this is exactly what we would expect as this important control demonstrates that the loss of monomeric perforin bands (as seen above) is due to formation of membrane-bound SDS-resistant oligomers on the cell membrane, rather than due to a physical loss of perforin. Asn549Gln does not, however, phenocopy WT with respect to oligomerisation of the full length perforin protein.
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Minor points: Can authors show the PCRs for KHYG1 CRIsP/Cas perforin WT/KO clones? Please, clearly indicate the strategy and sequences for gene editing. We thank the reviewer for the suggestion. These clones were assessed for absent perforin expression by Western immunoblotting following which clones were sequenced to show gene disruption. The sequences for sgRNA used are provided in the Methods section. Sequencing of the disrupted region is now added to Figure EV3, demonstrating an out of frame gene interference within a coding sequence of PRF1 in all KHYG1 perforin alleles, at c.359 (this information is now added to the Methods section); this is consistent with the loss of perforin protein as shown in the original Figure EV3. In page 5, authors describe several 'shorter perforin molecules' than the one that terminates at Leu 542 as ending at Leu 543 and Gly 544. These seem to be too long to be shorter. Please, clarify. The Reviewer is right - this now reads, “Multiple minor species corresponding to shorter or longer perforin molecules with C-termini at Gln540, Met541, Leu543 or Gly544 were also present.”
2nd Editorial Decision
23 June 2017
Thank you for the submission of your revised manuscript to our editorial offices. We have now received the reports from the two referees that were asked to re-evaluate your study (you will find enclosed below). As you will see, referee #2 fully supports the publication of your manuscript in EMBO reports, and also states your response to the other referee comments is adequate. Referee #3 has still concerns, and feels that further revisions are necessary. Considering the assessment of referee #2, we would ask you to add the quantification to the Western blots (point 6), and respond to the remaining points with text changes, or in the point-by-point-response. Further, I have the following editorial requests that need to be addressed in the final revised version: Please add up to 5 keywords and a running title to the title page. Please move the author contributions after the methods section, and add there a conflict of interest statement and acknowledgements (if you have acknowledgements). Please format the references according to EMBO reports style. Fig. EV1 seems to be a table. Please name it "Table EV1" and update its callout/s in the manuscript text. Please provide this table without color (our publisher does not allow tables in color), and then also update the remaining EV fig names (Fig. EV2 will then be EV1) and their callouts in the text. We would like to publish the paper as Scientific Report. For a Scientific Report we require that results and discussion are combined in a single chapter called "Results & Discussion". Please do that for your manuscript. Further, we allow up to five figures for a Scientific Report. Please combine panels of the existing figures in a way that we have 5 more compact figures in the end. Please refer to: http://embopress.org/sites/default/files/EMBOPress_Figure_Guidelines_061115.pdf Please add page numbers to the Appendix TOC. Appendix Figures should be named Appendix Fig. S1 and S2. Please add the "S" and change the callouts in the manuscript text accordingly. We now strongly encourage the publication of original source data with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure or per figure panel. I look forward to seeing a revised version of your manuscript when it is ready.
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REFEREE REPORTS Referee #2: The authors have addressed my comment and I feel that this provides some insight into the size of glycan necessary to inhibit perforin polymerisation in this context. The response to the other reviewer looks reasonable to me. Referee #3: Dear Authors, I thank you for taking the time and consideration to analyze and answer the previous comments on your manuscript. The work performed to obtain the piece of evidence shown herein with the C-terminal mutants by biochemical and molecular biology techniques is exhaustive and, of course merits consideration. However, as part of cell biology interest, the observation of intracellular localization is relevant. Even if you have performed similar experiments before, it can be that those results cannot be extrapolated to different mutants. Of course, there is no need to analyze all the mutants. Since you stated that your aim was to 'define a possible site for perforin processing by a granule-bound endopeptidase', one can hope that some illustrative images will appear. With regard to the lack of a consensus site for cleavage, the fact is that there is cleavage (of course), but accumulation of several species in the processing of a unique mutant might indicate a kind of requirement, that could be of interest, for cleavage (in Figure 2D there is a similar amount of 541 and 543 cleaved PRF and in Figure 2E same applies for 542 and 543, apart from the W555; all in red dotted line, in contrast with the black line representing WT PRF, with a unique/major peak at 542). For sure, it does not seem a big problem for processing. Indeed, a specific site for protease binding, different from the one for cleavage might exist in PRF, being dependent more on PRF folding than in the specific sequence of the C-terminus. It can be of interest to comment on this, if authors consider it maybe of relevance. With respect to model shown in Figure 3, it would be of interest for readers to have a more extended information on the program or application used to generate it, a reference to assess data used, etc. PBD ID 3NJS: Crystal structure of the complex formed between typeI ribosome inactivating protein and lactose at 2.1A resolution. I do not know if the search performed was correct. The direct comparison of WT, Asn549Gln and both PRFs purified upon treatment with E64D would allow to observe the possible differences or similarities in % of specific lysis of all the proteins at the same time (Fig 4F and 5B). The information on the amount of protein needed to obtain 50% specific lysis is important to see that Asn549Gln kills as much as WT (their processing is essentially similar, but for the unknown amount of residual flPRF). When in reference 19 Cytotoxic lymphocytes deficient in PRF were reconstituted with the Asn549Gln, their ability to kill target cells (EL4) were restored to a smilar extent than when rescued with the WT protein, in contrast with the mutant lacking both the 205 and 549 glycosylation sites (Figure 5B). Reconstitution of this latter mutant with a novel glycosylation site (not at the Cterminus) recovered the function of the double mutant in these PRF-deficient CTLs. A reader can ask why these CTLs only expresssing the Asn549Gln do not die by toxicity. And why they kill so much (Figure 6). And why a glycosylation site independent of the C-terminus can work as well as this one. These are points of interest to be included in the discussion. I actually understand that authors state from data shown on Figure 5D-F that, in the case of WT, flPRF is not decreased in Ca presence (20 min) with respect to 0 min. And that for Asn549Gln both prPRF and flPRF disappear. With the data shown, I have to disagree; it seems that flPRF decreases in both cases. There is less flPRF in the case of the mutant (0 min vs WT), and the reduction seems similar at 20 min (not the absolute amount or PRF). I mean, due to differences in the intensities of protein bands, I would say that I observe nearly equal decrease for flPRF in WT than in Asn549GLn
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mutant (from 0 to 20 min). However, I cannot asseverate that this effect is not due to resolution, contrast, or other causes. It is my impression that in CNCA there maybe also a subtle reduction from 0 to 20 min (+Ca). I really ask authors to quantify data from the three independent experiments performed and to show ratios for WT DMSO, CNCA and Asn549Gln. I know quantification is not an easy concept in western blot. But this makes difficult the analysis of the data when restrained differences are to be observed. Independently of their biological relevance. Indeed, authors have already performed it to quantify PRF immunoblot intensities with ImageJ in Cr-release assays. In the case the problem is the low amount of flPRF obtained (I have observed it is nearly null in most of the westerns shown for the Asn549Gln mutant), maybe an experiment of both WT and Asn549Gln pre-treated with E64D can be performed to increase flPRF content. I guess an important point can be the amount of the prPRF of the mutant that does not 'disappear' (20 min), which can be part of the mechanism (it can be displaced by the flPRF). I think this would really help the revision and also, the future readers of the work.
2nd Revision - authors' response
25 June 2017
1) The work performed to obtain the piece of evidence shown herein with the C-terminal mutants by biochemical and molecular biology techniques is exhaustive and, of course merits consideration. However, as part of cell biology interest, the observation of intracellular localization is relevant. Even if you have performed similar experiments before, it can be that those results cannot be extrapolated to different mutants. Of course, there is no need to analyze all the mutants. Since you stated that your aim was to 'define a possible site for perforin processing by a granule-bound endopeptidase', one can hope that some illustrative images will appear. If there were substantial differences in the cleavage sites of the various mutants, we agree that comparing localization would be important, but that is not the case. Even if we did wish to do the experiment (to generate “illustrative images”), it is virtually impossible as the cells already express three fluorescent proteins – GFP and Cherry from Cas9 and the guide, and BFP from perforin expression vector. Starting again from scratch would mean several months work for no actual gain. For the reasons stated below, we do not agree that this is necessary or desirable. The Reviewer fails to acknowledge that the observation of perforin cleavage in the granules is extremely well-documented by others (eg Uellner, 1997) and by us (eg Brennan, 2011). It has been shown that failure to traffic to the granules invariably results in failure of perforin to be cleaved. Below is an example from our paper (Chia, Blood, 2012) showing that failure of perforin mutant to traffic results in failure to be processed and, as a result, leads to the loss of NK cell function.
Using EndoH sensitivity as a marker of protein trafficking through the Golgi is universally accepted in the field, and has been used in dozens of papers. Furthermore, we have shown (Brennan, Immunity 2011) that even perforin mutants that have significantly delayed export from the endoplasmic reticulum, still localize in the granules at a steady-state level, yet perforin does not get cleaved until it reaches the granule compartment. In the current paper, we have shown using mass
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spectrometry that all of the critical mutants (Figure 2A-F) are cleaved in the same manner as the wild-type protein. All of the above has been explained in the manuscript. The notion of having “illustrative images” is anathema to us. We only wish to include robust, reproducible and original data, that actually add to the body of knowledge. The Reviewer has failed to explain the need for the additional work. 2) With regard to the lack of a consensus site for cleavage, the fact is that there is cleavage (of course), but accumulation of several species in the processing of a unique mutant might indicate a kind of requirement, that could be of interest, for cleavage (in Figure 2D there is a similar amount of 541 and 543 cleaved PRF and in Figure 2E same applies for 542 and 543, apart from the W555; all in red dotted line, in contrast with the black line representing WT PRF, with a unique/major peak at 542). For sure, it does not seem a big problem for processing. Indeed, a specific site for protease binding, different from the one for cleavage might exist in PRF, being dependent more on PRF folding than in the specific sequence of the C-terminus. It can be of interest to comment on this, if authors consider it maybe of relevance. We have commented on this in the text (lines 1-2, page 12) 3) With respect to model shown in Figure 3, it would be of interest for readers to have a more extended information on the program or application used to generate it, a reference to assess data used, etc. PBD ID 3NJS: Crystal structure of the complex formed between type I ribosome inactivating protein and lactose at 2.1A resolution. I do not know if the search performed was correct. We apologize for the typo – the correct PBD ID is 3NSJ, not 3NJS. 4) The direct comparison of WT, Asn549Gln and both PRFs purified upon treatment with E64D would allow to observe the possible differences or similarities in % of specific lysis of all the proteins at the same time (Fig 4F and 5B). The information on the amount of protein needed to obtain 50% specific lysis is important to see that Asn549Gln kills as much as WT (their processing is essentially similar, but for the unknown amount of residual flPRF). As we stated in our previous letter of rebuttal, the critical point is that the C-terminal glycosylation is inhibitory for perforin function, and we have shown it in many different ways throughout the manuscript. Despite our request (in the original rebuttal) to clarify a scientific reason for their query, i.e. what difference does it make whether WT has more or less activity than Asn549, the Reviewer has simply repeated their question. As we stated in the original rebuttal (and demonstrated throughout the paper), our experiments clearly show that E64D inhibition of the processing of the Cterminus of WT perforin leads to loss of function, due to the remaining glycan, whereas similar E64D inhibition of Asn549Gln processing has no effect on function because there is no glycan (Figures 3, 4 and Appendix Figure S2). What the Reviewer is essentially asking us to do (i.e. to repeat all of the purification experiments at least in triplicate - an extremely challenging and a time-consuming task) is superfluous and will not influence the conclusion of our work. 5) When in reference 19 Cytotoxic lymphocytes deficient in PRF were reconstituted with the Asn549Gln, their ability to kill target cells (EL4) were restored to a similar extent than when rescued with the WT protein, in contrast with the mutant lacking both the 205 and 549 glycosylation sites (Figure 5B). Reconstitution of this latter mutant with a novel glycosylation site (not at the Cterminus) recovered the function of the double mutant in these PRF-deficient CTLs. A reader can ask why these CTLs only expressing the Asn549Gln do not die by toxicity. And why they kill so much (Figure 6). And why a glycosylation site independent of the C-terminus can work as well as this one. These are points of interest to be included in the discussion. With respect, we do not understand the relevance of this question. Nonetheless, we would like to clarify that the issue of Asn549Gln toxicity has been already addressed in the current manuscript (please see pages 8, 10 and 12, highlighted).
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6) I actually understand that authors state from data shown on Figure 5D-F that, in the case of WT, flPRF is not decreased in Ca presence (20 min) with respect to 0 min. And that for Asn549Gln both prPRF and flPRF disappear. With the data shown, I have to disagree; it seems that flPRF decreases in both cases. There is less flPRF in the case of the mutant (0 min vs WT), and the reduction seems similar at 20 min (not the absolute amount or PRF). I mean, due to differences in the intensities of protein bands, I would say that I observe nearly equal decrease for flPRF in WT than in Asn549GLn mutant (from 0 to 20 min). However, I cannot asseverate that this effect is not due to resolution, contrast, or other causes. It is my impression that in CNCA there maybe also a subtle reduction from 0 to 20 min (+Ca). I really ask authors to quantify data from the three independent experiments performed and to show ratios for WT DMSO, CNCA and Asn549Gln. I know quantification is not an easy concept in western blot. But this makes difficult the analysis of the data when restrained differences are to be observed. Independently of their biological relevance. Indeed, authors have already performed it to quantify PRF immunoblot intensities with ImageJ in Crrelease assays. In the case the problem is the low amount of flPRF obtained (I have observed it is nearly null in most of the westerns shown for the Asn549Gln mutant), maybe an experiment of both WT and Asn549Gln pre-treated with E64D can be performed to increase flPRF content. I guess an important point can be the amount of the prPRF of the mutant that does not 'disappear' (20 min), which can be part of the mechanism (it can be displaced by the flPRF). We have now quantified the Western blots as requested (please see new Figure 4D-F and Figure Legend on page 20), and confirmed our findings. With respect to the rest of the query, we still do not fully understand, what the Reviewer is trying to say, but offer the following comment: The statement “In the case the problem is the low amount of flPRF obtained (I have observed it is nearly null in most of the westerns shown for the Asn549Gln mutant)…” is simply incorrect –the amount of flPRF in Asn549Gln mutant (please, see upper band at 0’ +Ca in F, below) is certainly comparable to WT Prf (shown in D).
We can only conclude that in their argument regarding WT perforin (see D above), the Reviewer is failing to understand our basic finding: the bottom band that represents cleaved (active) perforin disappears disproportionately more than the upper band in the presence of Ca (see the enlarged section of D on the right).
3rd Editorial Decision
27 June 2017
I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
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