Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public review): Summary:
The authors demonstrate that two human preproprotein human mutations in the BMP4 gene cause a defect in proprotein cleavage and BMP4 mature ligand formation, leading to hypomorphic phenotypes in mouse knock-in alleles and in Xenopus embryo assays.
Strengths:
They provide compelling biochemical and in vivo analyses supporting their conclusions, showing the reduced processing of the proprotein and concomitant reduced mature BMP4 ligand protein from impressively mouse embryonic lysates. They perform excellent analysis of the embryo and post-natal phenotypes demonstrating the hypomorphic nature of these alleles. Interesting phenotypic differences between the S91C and E93G mutants are shown with excellent hypotheses for the differences. Their results support that BMP4 heterodimers act predominantly throughout embryogenesis whereas BMP4 homodimers play essential roles at later developmental stages.
Weaknesses:
(1) A control of BMP7 alone in the Xenopus assays seems important to excludeBMP7 homodimer activity in these assays.
We and other have shown that BMP7 homodimers have weak or no activity while BMP4/7 heterodimers single at a much higher level than either BMP4 or BMP7 homodimers in Xenopus ectodermal and mesodermal cells. We have expanded the description of these published findings in the results section (lines 182-187). We have also added representative examples of experiments in which BMP4 and BMP7 alone controls are included (new Fig. S2). Since the level of activity of BMP7 + BMP4 variants is equivalent to that of BMP7 + WT BMP4, this cannot be accounted for by BMP7 homodimers.
(2) The Discussion could be strengthened by more in-depth explanations of how BMP4 homodimer versus heterodimer signaling is supported by the results, so that readers do not have to think it all through themselves. Similarly, a discussion of why the S91C mutant has a stronger phenotype than E93G early in the Discussion would be helpful or least mention that it will be addressed later.
We have revised the discussion as suggested by the reviewer. Please see responses to recommendations 2-4 below.
Reviewer #1 (Recommendations for the authors):
(1) A control of BMP7 injection alone seems missing when comparing the BMP4/7 variants. BMP4 in the embryo assays presented in Fig 1. Is it not possible that the activity observed is BMP7 homodimers, perhaps due to inhibited heterodimer formation by the BMP4 variant?
Multiple published studies have shown that BMP7 homodimers have weak or no activity in Xenopus ectodermal and mesodermal cells, and that ½ dose of RNA encoding BMP4 and BMP7 together signals at a higher level than does a full dose of RNA encoding either BMP4 or BMP7 alone. We have expanded our description of these published findings (lines 182-187), have included additional details about RNA doses that were injected (line 156, 175, 182) and have added representative examples of experiments in which BMP4 and BMP7 controls were included in a new Figure (Fig. S2).
(2) In reading the Discussion, I was continually thinking of the stronger phenotype of the S91C mutant compared to the E93G one, although both are discussed together throughout most of the Discussion. Only at the end of the Discussion is the stronger phenotype of S91C discussed with a compelling explanation for the stronger phenotype, not related to the phosphorylation site function. I wonder if it would be better placed earlier in Discussion or at least mentioned the difference in phenotypes that will be discussed later.
We have moved the possible explanation of differences between Bmp4S91C and Bmp4E93G mutants to immediately follow the introductory paragraph of the results section.
(3) Along these same lines, why is it that the E93G exhibits rather normal cleavage at E10.5? Might the mechanisms of cleavage vary in different contexts with phosphorylation-dependent cleavage not functioning at early stages of development? I believe the hypothesis is that it is cleaved due to heterodimerization with BMP7. More discussion of this excellent hypothesis should be provided with clear statements, rather than inferences, if I'm understanding this correctly. For example, I had to read 3 times the first sentence of the last paragraph on p.14 before I understood it. Better to break that sentence down and the one that follows it, so it is easier to understand.
We have rewritten and expanded the paragraphs describing phenotypic and biochemical evidence for defective homodimer but not heterodimer signaling as suggested (lines 343-375). We have also more explicitly stated the possibility that normal cleavage of BMP4E93G in embryonic lystates may be due to a predominance of BMP4/7 heterodimers in early embryonic stages or spatiotemporal differences in phosphorylation-dependent cleavage of BMP4 homodimers (lines 369-372)
(4) Similarly the last paragraph of the Discussion mentions that the authors provide evidence of BMP4 homodimer signaling. I agree with the authors, but I had to think through the evidence myself. Better if the authors clearly explain the evidence that points to this, as this is a very good point of
See response to point 3, above. Thank you for these useful suggestions.
(5) Last sentence, first paragraph on p.11 should be qualified for the E93G mutant to E13.5, since it was normal at E10.5 regarding Figure 4 results.
Thank you for pointing this out. It has been corrected.
(6) Skip the PC acronym, since it is only repeated once in the text and hard to remember almost 10 pages later when it is used again.
We have corrected this.
(7) In the Discussion, a typo in "a single intramolecular disulfide bond that stabilizes the dimer", should be 'intermolecular'.
Thank you for catching our switch in the use of inter- and intramolecular. We have corrected this (lines 334-335).
(8) At times the E93G mutant is referred to having early lethality, often in conjunction with S91C, while other times it is referred to as late lethality. Considering that the homozygotes die postnatally after weaning, most would consider it late lethality. In contrast S91C is indeed an early lethal.
We have changed the wording in the introduction to state that “mice carrying Bmp4S91C or Bmp4E93G knock in mutations show embryonic or enhanced postnatal lethality, respectively,… (lines 141-143)” and have removed the word “early” from the title.
Reviewer #2 (Public review): Summary:
Kim et al. report that two disease mutations in proBMP4, Ser91Cys and Glu93Gly, which disrupt the Ser91 FAM20C phosphorylation site, block the activation of proBMP4 homodimers. Consequently, analysis of DMZ explants from Xenopus embryos expressing the proBMP4 S91C or E93G mutants showed reduced pSmad1 and tbxt1 expression. The block in BMP4 activity caused by the mutations could be overcome by co-expression of BMP7, suggesting that the missense mutations selectively affect the activity of BMP4 homodimers but not BMP4/7 heterodimers. The expert amphibian tissue transplant studies were extended to in vivo studies in Bmp4S91C/+ and Bmp4E93G/+ mice, demonstrating the impact of these mutations on embryonic development, particularly in female mice, in line with patient studies. Finally, studies in MEFs revealed that the mutations did not affect proBMP4 glycosylation or ER-to-Golgi transport but appeared to inhibit the furin-dependent cleavage of proBMP4 to BMP4. Based on these findings and AI (AlphaFold) modeling of proBMP4, the authors speculate that pSer91 influences access of furin to its cleavage site at Arg289AlaLysArg292.
Strengths:
The Xenopus and mouse studies are valuable and elegantly describe the impact of the S91C and E93G disease mutations on BMP signaling and embryonic development.
Weaknesses:
The interpretation of how the mutations may disturb the furin-mediated cleavage of proBMP4 is underdeveloped and does not consider all of their data. Understanding how pS91 influences the furin-dependent cleavage at Arg292 seems to be the crux of this work and thus warrants more consideration. Specifically:
(1) Figure S1 may be significantly more informative than implied. The authors report that BMP4S91D activates pSmad1 only incrementally better than S91C and much less than WT BMP4. However, Fig. S1B does not support the conclusion on page 7 (numbering beginning with title page); "these findings suggest that phosphorylation of S91 is required to generate fully active BMP4 homodimers". The authors rightly note that the S91C change likely has manifold effects beyond inhibiting furin cleavage. The E93G change may also affect proBMP4 beyond disturbing FAM20C phosphorylation. Additional mutation analyses would strengthen the work.
The major goal of generating and comparing the activity of the S91D mutant with S91C was to control for phosphorylation independent defects cause by the deleterious introduction of a cysteine residue, which might cause aberrant disulfide bonding. We opted to introduce S91D since “phosphomimics” can sometimes approximate the phosphorylated state. S91D has significantly higher activity than S91C (p<0.01) and has a less significant loss of activity (p<0.05) than does S91C (<p<0.0001) relative to wild type BMP4 (Fig. S1), consistent with deleterious effects of the cysteine residue and supporting a possible explanation for the more severe phenotype of S91C vs E93G mice. We have rewritten this section to clarify our interpretation (lines 165-174)and have changed our statement that our activity data “suggest the importance of phosphorylation” to a statement that they are consistent with this possibility (lines 179-180). We do not believe that further mutational analysis using activity assays in Xenopus would shed light on how or whether phosphorylation affects proteolytic activation of BMP4.
(2) These findings in Figure S1 are potentially significant because they may inform how proBMP4 is protected from cleavage during transit through the TGN and entry into peripheral cellular compartments. Intriguing modeling studies in Figure 6 suggest that pSer91 is proximal to the furin cleavage site. Based on their presentation, pSer91 may contact Arg289, the critical P4 residue at the furin site. If so, might that suggest how pS91 may prevent furin cleavage, thus explaining why the S91D mutation inhibits processing as presented, and possibly how proBMP4 processing is delayed until transit to distal compartments (perhaps activated by a change in the endosomal microenvironment or a Ser91 phosphatase)? Have the authors considered or ruled out these possibilities? In addition to additional mutation analyses of the FAM20C site, moving the discussion of this model to an "Ideas and Speculation" subsection may be warranted.
The model shown in Fig. 6B proposes the possibility that phosphorylation unmasks (rather than preventing) the furin cleavage motif due to the proximity of Ser91 to the cleavage site (lines 399-402). If S91D truly mimicked phosphorylation, we would predict it would facilitate processing rather than inhibiting it. We do not have data comparing cleavage of S91D relative to wild type BMP4 and have not generated knock in S91D mice to test this idea. While the reviewers questions are intriguing, they cannot be answered by mutational analysis of the FAM20C site and are beyond the scope of the current studies that sought to understand the impact of human pS91C and pE93G mutations and cell biological implications. We have moved the models to an “Ideas and Speculation” subsection as suggested (lines 377-414) since these models are meant to provoke further thought rather than provide definitive answers based on our data.
(3) The lack of an in vitro protease assay to test the effect of the S91 mutations on furin cleavage is problematic.
Although we routinely perform in vitro cleavage assays with recombinant furin, we don’t believe they would be informative on how S91 phosphorylation or mutation of this residue impacts cleavage since in vitro synthesized substrate used in these assays is neither dimerized not post-translationally modified, and cleavage would be tested in isolation from the endogenous trafficking environment that we propose influences cleavage.
Reviewer #2 (Recommendations for the authors):
(1) The impact of BMPS91A should be determined and paired with the S91D phosphomimic data to reveal if it causes proBMP4 to be cleaved prematurely and disturbs pSmad1 expression. Data for S93G should also be included.
Our major goal in comparing the activity of S91D with S91C was to control for phosphorylation independent defects cause by the deleterious introduction of a cysteine residue in S91C, which might cause aberrant disulfide bonding. We opted to introduce S91D since “phosphomimics” can sometimes approximate the phosphorylated state. We note that S91D has significantly higher activity than S91C, consistent with deleterious effects of the cysteine residue and supporting a possible explanation for the more severe phenotype of S91C vs E93G mice. We have revised the wording of this section to clarify this. Our models predict that S91D would be cleaved more efficiently than S91C or S91A, if it really mimics the endogenous phosphorylated state, rather than being cleaved prematurely. Our biochemical analysis compares cleavage of endogenous BMP4 in wild type and mutant MEFs. Generation of S91D, S91A or S93G mutant mice to compare cleavage is beyond the scope of the current work.
(2) Is the distance between pS91 and Arg289 close enough to form a hydrogen bond? If so, might this interaction influence furin access?
AI modeling does not provide high probability prediction of structures surrounding the furin motif (see Fig. S7) and thus we cannot comment on whether or not these residues are close enough to form a hydrogen bond. We have revised the wording of the discussion to state “This simple model building indicates the possibility of direct contact between pSer91 and Arg289, and that phosphorylation is required for furin to access the cleavage site, although we note that predictions surrounding the furin motif represent low probability conformations (Fig. S7) (lines 399-402).”
(3) The genotypes in Figure 2 are labeled awkwardly. Consider labeling the headers for the three subsections of panels (A-F, G-L, and M-O) differently.
We have revised Fig. 2 to clarify that the three subsections of panels are distinct, and to emphasize that the middle subsection represents views of the right and left side of the same embryo.
(4) The tables should be reformatted. As is, the labeling is frequently cut off, and the numbers of expected and observed progeny should both be stated to aid the reader.
We thank the reviewer for noting the formatting errors in the tables, which we have corrected. We have also changed the tables so that normal or abnormal mendelian distributions are reported as numbers of observed/expected progeny rather than numbers/percent observed progeny.
Reviewer #3 (Public review):
Summary:
The authors describe important new biochemical elements in the synthesis of a class of critical developmental signaling molecules, BMP4. They also present a highly detailed description of developmental anomalies in mice bearing known human mutations at these specific elements.
Strengths:
Exceptionally detailed descriptions of pathologies occurring in mutant mice. Novel findings regarding the interaction of propeptide phosphorylation and convertase cleavage, both of which will move the field forward. Provocative hypothesis regarding furin access to cleavage sites, supported by Alphafold predictions.
Weaknesses:
Figure 6A presents two testable models for pre-release access of furin to cleavage sites since physical separation of enzyme from substrate only occurs in one model; could immunocytochemistry resolve?
Available reagents are not sensitive enough to detect endogenous furin and BMP4 with high resolution. Because PC/substrate interactions are transient, whereas the bulk of furin and BMP4 is distributed throughout the secretory pathway, it is not possible to co-immunolocalize furin and BMP4 in vivo at present. Studies using more advanced cell biological techniques such along with tagged proteins may enable us to test these hypotheses in the future.
Reviewer #3 (Recommendations for the authors):
This interesting paper presents new data on an important family of developmental signaling molecules, BMPs. Mutations at FAM20C consensus sites within BMP prodomains are known to cause birth defects. The authors have here explored differential effects of human mutations on hetero- and homodimer activity and maturation, issues that may well arise during human development. In addition to demonstrating the profound effect of these mutations on development in Xenopus and mice, the authors also show differential processing of BMP4 precursors bearing these mutations in MEF cells prepared from mutant embryos. Finally, they show that FAM20C plays a role in BMP4 prodomain processing with quite differing outcomes in homo- vs heterodimers, which they suggest is due to structural differences impacting furin access. While this latter idea remains speculative due to the lack of crystal structures (models are based on Alphafold) it is a highly promising line of work.
The data are beautifully presented and will be of clear interest to all developmental biologists. Certain cell biology results may also extrapolate to other phosphorylated precursor molecules undergoing the interesting (and as yet unexplained) phenomenon of convertase cleavage immediately before secretion, for example, FGF23. I have only a few minor comments regarding the presentation, which is remarkably clear.
(1) The introduction of BMP7 in the Abstract is abrupt. It should be described as a preferred dimerization partner for BMP4.
Thank you for noting this. We have revised the first sentence of the abstract to better introduce BMP7(lines 49-50).
(2) In Figure 1A, what is the small light green box?
This is a small fragment released from the prodomain by the second cleavage. We have clarified this in the introduction (lines 112-114) and in the legend to Figure 1 (lines 758-759).
(3) In the Discussion it might be relevant to mention that FAM20C propeptide is not cleaved by convertases but by S1P (Chen 2021).
We have added this information to clarify (lines 394-396).
(4) Figure 3, define VSD; Figure 5, Endo H removes sugars only from immature (nonsialylated) sugars, not from all chains as implied. More importantly, EndoH and PNGase remove N-linked sugars, yet Results refer only to O-linked glycosylation.
Thank you for noting these oversights. We have defined VSD in Figure 3. We have also revised the headers for Fig. 5 and for the relevant subsection of the results to include N-linked glycosylation and note in the results that EndoH removes only immature N-linked carbohydrates (lines 301-304).
(5) Figure 5- for clarity, I suggest it be broken up into two larger panels labeled "Embryos" and "MEFs"
Thank you for this suggestion, we have subdivided the Figure into two panels.
(6) Figure 6A presents two testable models for pre-release access of furin to cleavage sites since the physical separation of the enzyme from substrate only occurs in one model; could confocal immunocytochemistry resolve?
Available reagents are not sensitive enough to detect endogenous furin and BMP4 with high resolution and PC/substrate interactions are transient whereas the bulk of both furin and BMP4 is in transit through the secretory pathway. For these reasons it is not possible to co-immunolocalize furin and BMP4 in vivo. Future studies using advanced cell biological techniques may enable us to test these hypotheses in the future.
