Glypican-1 drives unconventional secretion of fibroblast growth factor 2
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Evaluation Summary:
FGF2 moves directly from the cytoplasm through the plasma membrane in a reaction driven by its subsequent high affinity binding to cell surface heparan sulfate proteoglycans. This study concludes that Glypican-1 is the principal proteoglycan involved, possibly involving a unique tri-sulfated disaccharide binding site in close proximity to the cell surface. While the role of Glypican-1 appears unique to FGF2 rather than part of a generalized direct secretion mechanism, the observations highlight the complexity and significance of proteoglycan variation. The work is well done and generally convincing, but additional support for the authors' conclusion that a specific glycan structure in GPC1 is a specific ligand for FGF2 is required.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse subclasses consisting of syndecans, perlecans, glypicans, and others, Glypican-1 (GPC1) is the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.
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Evaluation Summary:
FGF2 moves directly from the cytoplasm through the plasma membrane in a reaction driven by its subsequent high affinity binding to cell surface heparan sulfate proteoglycans. This study concludes that Glypican-1 is the principal proteoglycan involved, possibly involving a unique tri-sulfated disaccharide binding site in close proximity to the cell surface. While the role of Glypican-1 appears unique to FGF2 rather than part of a generalized direct secretion mechanism, the observations highlight the complexity and significance of proteoglycan variation. The work is well done and generally convincing, but additional support for the authors' conclusion that a specific glycan structure in GPC1 is a specific ligand for FGF2 is required.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
FGF2 moves directly from the cytoplasm through the plasma membrane in a reaction driven by its subsequent high affinity binding to cell surface heparan sulfate proteoglycans. This study concludes that Glypican-1 is the principal proteoglycan involved, possibly involving a unique tri-sulfated disaccharide binding site in close proximity to the cell surface. While the role of Glypican-1 appears unique to FGF2 rather than part of a generalized direct secretion mechanism, the observations highlight the complexity and significance of proteoglycan variation. The work is well done and generally convincing, but additional support for the authors' conclusion that a specific glycan structure in GPC1 is a specific ligand for FGF2 is required.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this paper, gene deletions, complementations, and careful phenotype analyses define roles of GPC1 in secretion and binding to the autocrine cell surface. GPC1 which is GPI-anchored to the cell surface, facilitates export of FGF2, acts as a highly specific surface receptor for FGF2. GPC1 is specific in these activities, i.e. it is much more active than other heparan sulfate-containing GAGs. A chimeric protein with the GPC1 N-terminal and the transmembrane segment of another GAG can substitute for GPI-anchored GPC1. GPC1 is dispensable for FGF2 downstream signaling. Thus, this work describes specific molecular roles for the GPC1 extracellular region in the lab's long-standing investigation of the unconventional secretion mechanisms for FGF2. A disaccharide analysis leads to the proposal that the specificity …
Reviewer #1 (Public Review):
In this paper, gene deletions, complementations, and careful phenotype analyses define roles of GPC1 in secretion and binding to the autocrine cell surface. GPC1 which is GPI-anchored to the cell surface, facilitates export of FGF2, acts as a highly specific surface receptor for FGF2. GPC1 is specific in these activities, i.e. it is much more active than other heparan sulfate-containing GAGs. A chimeric protein with the GPC1 N-terminal and the transmembrane segment of another GAG can substitute for GPI-anchored GPC1. GPC1 is dispensable for FGF2 downstream signaling. Thus, this work describes specific molecular roles for the GPC1 extracellular region in the lab's long-standing investigation of the unconventional secretion mechanisms for FGF2. A disaccharide analysis leads to the proposal that the specificity and affinity of GPC1 for FGF2 is dependent on the increased concentration of ligand specific to GPC1. However, the data supporting this idea is not is not convincing. Nevertheless, the further elucidation of the mechanism of non-conventional secretion is of interest to a broad readership in cancer, and glycobiology, and cell biology. The work is carefully described in appropriate detail.
The authors propose that the increased presence of a specific disaccharide sequence in GPC1 explains the high specificity and affinity relative to other heparan sulfate GAGs. However, the modest 1.2-fold increase of this disaccharide is insufficient to explain the difference in FGF2 affinity. Therefore, the manuscript needs to be revised to accommodate other possible explanations for the great specificity and affinity of GPC1 as the receptor and export facilitator.
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Reviewer #2 (Public Review):
This is a rigorously designed and carefully controlled study that is well-presented. I have only the following issues.
1. The authors raise the issue of how BirA might be able to label GP1 given the ATP requirement. Is this simply that FGF2-BirA is bound to the activated biotin as it transits the membrane? Are the kinetics of FGF2 transfer consistent with the stability of this intermediate of BirA?
2. Fig 2 shows a strong reduction of FGF2 secretion upon GPC1 KO using the surface biotinylation assay (approx. 75% with little error). This is the critical evidence that GPC1 is required, yet in Fig4A and D the result looks much less convincing (50% with large error). For Fig4D the WT data are present and it seems questionable whether there is a significant difference. This needs to be explained and/or corrected. …Reviewer #2 (Public Review):
This is a rigorously designed and carefully controlled study that is well-presented. I have only the following issues.
1. The authors raise the issue of how BirA might be able to label GP1 given the ATP requirement. Is this simply that FGF2-BirA is bound to the activated biotin as it transits the membrane? Are the kinetics of FGF2 transfer consistent with the stability of this intermediate of BirA?
2. Fig 2 shows a strong reduction of FGF2 secretion upon GPC1 KO using the surface biotinylation assay (approx. 75% with little error). This is the critical evidence that GPC1 is required, yet in Fig4A and D the result looks much less convincing (50% with large error). For Fig4D the WT data are present and it seems questionable whether there is a significant difference. This needs to be explained and/or corrected. Statistical significance of WT vs KO should be reported in Fig1 and the result should be reproducible throughout, if using the same assay.
3. The authors imply that FGF2 binding to proteoglycans is critical for it to initiate signaling and their data argue that this does not require GPC1. The role of proteoglycans here was confusing to me and may be to other readers. The authors should tell us how proteoglycan binding relates to RTK binding, and whether there is a reason that lower affinity interactions with proteoglycan sites (relative to the higher affinity interaction with GPC1) would be sufficient in this role. -
Reviewer #3 (Public Review):
The authors claim that GPC1, a conventionally secreted GPI anchored glycoprotein, binds unconventionally secreted FGF2 at the cell surface. Loss of GPC1 affects FGF2 secretion and over expression of GPC1 stimulates FGF2 secretion. The authors also identify the specific sugars with N-linked sulphate groups in GPC1 that appear to function in FGF2 secretion.The data are of high quality. Overall, the paper provides a new component in the pathway of unconventional secretion of FGF2. Additional data are necessary to rule out the trivial possibility that loss of GPC1 affects membrane properties that indirectly affect the translocation of FGF2.
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