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Glypicans shield the Wnt lipid moiety to enable signalling at a distance

Abstract

A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1,2,3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4,5,6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

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Fig. 1: Spread of Wingless does not involve multimeric assemblies but requires Dlp.
Fig. 2: A subset of glypicans bind palmitoleate.
Fig. 3: Structural basis of glypican-Wnt peptide interaction.
Fig. 4: Structure-guided point mutants impair Dlp lipid interaction.

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Data availability

X-ray crystallographic coordinates and structure factor files generated during the current study are available from the RCSB Protein Data Bank (PDB) under accession code 6XTZ. Full scans for all western blots are provided in Supplementary Fig. 1. Source data are provided with this paper.

References

  1. Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).

    CAS  PubMed  ADS  Google Scholar 

  2. Takada, R. et al. Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev. Cell 11, 791–801 (2006).

    CAS  PubMed  Google Scholar 

  3. Janda, C. Y., Waghray, D., Levin, A. M., Thomas, C. & Garcia, K. C. Structural basis of Wnt recognition by Frizzled. Science 337, 59–64 (2012).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  4. Panáková, D., Sprong, H., Marois, E., Thiele, C. & Eaton, S. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005).

    PubMed  ADS  Google Scholar 

  5. Gross, J. C., Chaudhary, V., Bartscherer, K. & Boutros, M. Active Wnt proteins are secreted on exosomes. Nat. Cell Biol. 14, 1036–1045 (2012).

    CAS  PubMed  Google Scholar 

  6. Mulligan, K. A. et al. Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility. Proc. Natl Acad. Sci. USA 109, 370–377 (2012).

    CAS  PubMed  ADS  Google Scholar 

  7. Kiecker, C. & Niehrs, C. A morphogen gradient of Wnt/β-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development 128, 4189–4201 (2001).

    CAS  PubMed  Google Scholar 

  8. Farin, H. F. et al. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche. Nature 530, 340–343 (2016).

    CAS  PubMed  ADS  Google Scholar 

  9. Alexandre, C., Baena-Lopez, A. & Vincent, J. P. Patterning and growth control by membrane-tethered Wingless. Nature 505, 180–185 (2014).

    CAS  PubMed  ADS  Google Scholar 

  10. Tian, A., Duwadi, D., Benchabane, H. & Ahmed, Y. Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization. PLoS Genet. 15, e1008111 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Zecca, M., Basler, K. & Struhl, G. Direct and long-range action of a wingless morphogen gradient. Cell 87, 833–844 (1996).

    CAS  PubMed  Google Scholar 

  12. Harmansa, S., Hamaratoglu, F., Affolter, M. & Caussinus, E. Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527, 317–322 (2015).

    CAS  PubMed  ADS  Google Scholar 

  13. Yan, D., Wu, Y., Feng, Y., Lin, S. C. & Lin, X. The core protein of glypican Dally-like determines its biphasic activity in wingless morphogen signaling. Dev. Cell 17, 470–481 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Franch-Marro, X. et al. Glypicans shunt the Wingless signal between local signalling and further transport. Development 132, 659–666 (2005).

    CAS  PubMed  Google Scholar 

  15. Stanganello, E. et al. Filopodia-based Wnt transport during vertebrate tissue patterning. Nat. Commun. 6, 5846 (2015).

    CAS  PubMed  ADS  Google Scholar 

  16. Baeg, G. H., Selva, E. M., Goodman, R. M., Dasgupta, R. & Perrimon, N. The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors. Dev. Biol. 276, 89–100 (2004).

    CAS  PubMed  Google Scholar 

  17. Reichsman, F., Smith, L. & Cumberledge, S. Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 135, 819–827 (1996).

    CAS  PubMed  Google Scholar 

  18. Baena-Lopez, L. A., Franch-Marro, X. & Vincent, J. P. Wingless promotes proliferative growth in a gradient-independent manner. Sci. Signal. 2, ra60 (2009).

    PubMed  PubMed Central  Google Scholar 

  19. Tang, X. et al. Roles of N-glycosylation and lipidation in Wg secretion and signaling. Dev. Biol. 364, 32–41 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kakugawa, S. et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187–192 (2015).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  21. Fuerer, C., Habib, S. J. & Nusse, R. A study on the interactions between heparan sulfate proteoglycans and Wnt proteins. Dev. Dyn. 239, 184–190 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Mihara, E. et al. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin. eLife 5, e11621 (2016).

    PubMed  PubMed Central  Google Scholar 

  23. Kim, M. S., Saunders, A. M., Hamaoka, B. Y., Beachy, P. A. & Leahy, D. J. Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling. Proc. Natl Acad. Sci. USA 108, 13112–13117 (2011).

    CAS  PubMed  ADS  Google Scholar 

  24. Pei, J. & Grishin, N. V. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins. Protein Sci. 21, 1172–1184 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hirai, H., Matoba, K., Mihara, E., Arimori, T. & Takagi, J. Crystal structure of a mammalian Wnt-frizzled complex. Nat. Struct. Mol. Biol. 26, 372–379 (2019).

    CAS  PubMed  Google Scholar 

  26. Awad, W. et al. Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1. J. Biol. Chem. 290, 22991–23008 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Sivasankaran, R., Calleja, M., Morata, G. & Basler, K. The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family. Mech. Dev. 91, 427–431 (2000).

    CAS  PubMed  Google Scholar 

  28. Schilling, S., Steiner, S., Zimmerli, D. & Basler, K. A regulatory receptor network directs the range and output of the Wingless signal. Development 141, 2483–2493 (2014).

    CAS  PubMed  Google Scholar 

  29. Mii, Y. & Taira, M. Secreted Frizzled-related proteins enhance the diffusion of Wnt ligands and expand their signalling range. Development 136, 4083–4088 (2009).

    CAS  PubMed  Google Scholar 

  30. Hayashi, Y., Kobayashi, S. & Nakato, H. Drosophila glypicans regulate the germline stem cell niche. J. Cell Biol. 187, 473–480 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang, X. & Page-McCaw, A. A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation. J. Cell Biol. 206, 923–936 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Serralbo, O. & Marcelle, C. Migrating cells mediate long-range WNT signaling. Development 141, 2057–2063 (2014).

    CAS  PubMed  Google Scholar 

  33. González-Méndez, L., Gradilla, A. C. & Guerrero, I. The cytoneme connection: direct long-distance signal transfer during development. Development 146, dev174607 (2019).

    PubMed  Google Scholar 

  34. Elegheert, J. et al. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nat. Protocols 13, 2991–3017 (2018).

    CAS  PubMed  Google Scholar 

  35. Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002).

    CAS  PubMed  ADS  Google Scholar 

  36. Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D 62, 1243–1250 (2006).

    PubMed  Google Scholar 

  37. Chang, V. T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D 61, 651–657 (2005).

    PubMed  Google Scholar 

  39. Newman, J. Novel buffer systems for macromolecular crystallization. Acta Crystallogr. D 60, 610–612 (2004).

    PubMed  Google Scholar 

  40. Winter, G., Lobley, C. M. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D 69, 1260–1273 (2013).

    CAS  PubMed  Google Scholar 

  41. Winter, G. et al. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr. D 74, 85–97 (2018).

    CAS  Google Scholar 

  42. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013).

    CAS  PubMed  Google Scholar 

  43. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  Google Scholar 

  44. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    CAS  PubMed  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  Google Scholar 

  47. Smart, O. S. Grade v.1.105; http://grade.globalphasing.org (2012).

  48. Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    CAS  PubMed  Google Scholar 

  49. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  PubMed  Google Scholar 

  50. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Tian, W., Chen, C., Lei, X., Zhao, J. & Liang, J. CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res. 46 (W1), W363–W367 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Port, F., Chen, H. M., Lee, T. & Bullock, S. L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc. Natl Acad. Sci. USA 111, E2967–E2976 (2014).

    CAS  PubMed  Google Scholar 

  54. Liebschner, D. et al. Polder maps: improving OMIT maps by excluding bulk solvent. Acta Crystallogr. D 73, 148–157 (2017).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Alexandre for plasmids and advice, J. Kurth for Drosophila injections, T. Walter for technical support with crystallization, and W. Lu and Y. Zhao for help with tissue culture and advice. We thank K. Harlos and staff at Diamond Light Source beamlines (i03, i04 and i04-1) for assistance with X-ray data collection (proposal MX19946). This work was supported by core funding to the Francis Crick Institute to J.P.V. (CRUK FC001204, MRC FC001204, and Wellcome Trust FC001204), the European Union (ERC grants WNTEXPORT (294523) to J.P.V., and CilDyn (647278) to C.S.), Cancer Research UK (C375/A17721 to E.Y.J., and C20724/A26752 to C.S.). L.V. is supported by Wellcome Trust PhD Training Programme 102164/B/13/Z. The Wellcome Centre for Human Genetics is supported by Wellcome Trust Centre grant 203141/Z/16/Z.

Author information

Authors and Affiliations

Authors

Contributions

Experimental contributions were as follows: Drosophila developmental genetics and cell-based assays (I.J.M.), genetic analysis of LPPs (K.B.), biophysics and structural biology (L.V., B.B., T.M., C.S.); peptide synthesis (D.J., N.O’R.). The project was conceived by I.J.M., L.V., E.Y.J. and J.-P.V. The first draft of the paper was written by J.-P.V., E.Y.J., I.J.M. and L.V. All the authors contributed to the design and interpretation of experiments.

Corresponding authors

Correspondence to E. Yvonne Jones or Jean-Paul Vincent.

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The authors declare no competing interests.

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Peer review information Nature thanks Elina Ikonen, Daniel Leahy, Roeland Nusse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Extracellular Wingless, captured by morphotrap, colocalizes with DlpΔGPI but not exosomes or lipophorin particles.

a, A membrane-tethered anti-GFP nanobody (Vhh4-CD8-HA, morphotrap), expressed in a transversal stripe with dpp-Gal4, leads to accumulation of GFP-Wingless (from a knock-in allele53) where the two expression domains overlap. The apparent gap in morphotrap expression is due to the known low activity of dpp-gal4 there. Residual expression is nevertheless sufficient to trap GFP-Wingless. bd, Immunofluorescent localization of morphotrap-enriched GFP–Wingless and endogenous ApoL or endogenous Hrs. d, Immunofluorescent localization of morphotrap-enriched GFP–Wingless and overexpressed DlpΔGPI–V5. Here, morphotrap is expressed with dpp-LexA and DlpΔGPI–V5 with wg-Gal4. White colour indicates extensive colocalization. DlpΔGPI–V5 expression was limited to 24 h with Gal80ts to avoid pleiotropic effects. e, Colocalization was quantified where the morphotrap and GFP–Wingless expression domains intersect. Error bars denote s.d. n = 4 (DlpΔGPI–V5), n = 13 (ApoL) and n = 10 (Hrs), in which n denotes number of wing discs. Scale bars, 50 μm (a), 10 μm (bd). All experiments were repeated independently three times with similar results.

Source data

Extended Data Fig. 2 Genetic perturbation of exosomes or lipophorin particles does not alter the distribution of extracellular Wingless.

a, Expression of dominant-negative Vps4 with hh-Gal4 in the posterior compartment (limited to 8 h with Gal80ts to avoid pleiotropic effects of sustained VPS4 inhibition) does not affect extracellular Wingless despite disruption of MVB formation indicated by accumulation of ubiquitin. Anterior compartment serves as a control. Dashed line denotes anterior posterior boundary. b, Expression of extracellular Wingless is largely unaffected by the loss of Hrs activity. The posterior compartment was rendered homozygous for a null hrs mutation using the indicated genotype. c, d, Overexpression of the lipophorin receptor Lpr2E–HA for 24 h with the hh-Gal4 driver increases the uptake of ApoL in the posterior compartment but has no effect on extracellular Wingless. The anterior compartment serves as a control. e, Extracellular Wingless and ApoL in wing discs from w118 (control) or homozygotes for a deficiency that removes the two main lipophorin receptors Lpr1/2. The uptake of lipophorin is reduced in the deficiency line but neither total nor extracellular Wingless is altered. Scale bars, 50 μm. All experiments were repeated independently three times with similar results.

Extended Data Fig. 3 Genetic perturbation of Dlp and dally but not of Swim alters the distribution of extracellular Wingless.

a, Expression of an RNAi against Swim in the posterior compartment (en-Gal4) does not alter extracellular or total Wingless. Scale bar, 50 μm. b, Schematic of the Swim locus and the CRISPR–Cas9 strategy used to delete the gene. Successful deletion was verified by PCR using two independent primer pairs indicated by purple and red arrowheads. c, Extracellular Wingless and Distal-less expression in control (w1118) and SwimKO wing discs. Neither is altered afteer Swim deletion. Scale bars, 50 μm. d, Extracellular Wingless is reduced in clones lacking both Dlp and Dally (marked by the absence of GFP (white arrowheads). Scale bars, 25 μm. All experiments were repeated independently three times with similar results.

Extended Data Fig. 4 Further evidence for the lipid binding activity of Dlp class glypicans.

a, GFP–Wingless(S239A) expressed from a knock-in allele is trapped by CD8-VHH expressed with dpp-Gal4. This result shows that GFP–Wingless(S239A) is secreted and can be captured in the extracellular space and thus serves as a positive control for Fig. 2a. Scale bars, 50 μm. b, Dlpcore and Notum(S237A), but not Dallycore, preferentially bind a palmitoleoylated peptide (sequence from the Wingless protein). Biotinylated palmitoleoylated and non-palmitoleoylated peptides were incubated with medium from S2 cells expressing Dlpcore–V5, Notum(S237A)–V5 or Dallycore–V5. Biotinylated peptides were pulled down with streptavidin beads and the extent to which Dlpcore, Notum(S237A)–V5 and Dallycore are co-pulled down was determined by western blot. A peptide-based approach was used because of difficulties associated with the production of soluble Wingless protein. Binding was estimated from the amount of protein co-pulled down from conditioned medium by streptavidin beads. c, Steady-state analysis of the Dlpcore versus palmitoleoylated human WNT7A peptide interaction measured by BLI shown in Fig. 2b. Kd is calculated from a global fit of three independent experiments. Owing to the effect of non-specific binding on the shape of the binding isotherm curves (which could not be alleviated by detergents in light of the lipid-based nature of the interaction), we could not perfectly fit a 1:1 Langmuir model, therefore the indicated apparent dissociation constant is an estimate, not an exact value. d, Representative reference-subtracted BLI traces of Dlpcore against biosensors loaded with non-palmitoleoylated peptide (sequence from relevant region of human WNT7A). No significant binding could be detected (compare to Fig. 2b). The experiments were repeated independently three times with similar results. e, Human GPC4 (Dlp family), but not human GPC3 (Dally family), expressed with ptc-Gal4 captured GFP–Wingless at the cell surface. This panel extends the data of Fig. 2c. Scale bars, 50 μm. All experiments were repeated independently three times with similar results.

Source data

Extended Data Fig. 5 Effects of Dlp class glypicans on Wingless signalling.

a, GPC4 and GPC6, but not GPC3 and GPC5, driven with ptc-Gal4 inhibit the high target gene senseless. Scale bars, 50 μm. b, GPC4 and GPC6, but not GPC3 and GPC5, expressed with en-Gal4 extend the range of the low target gene Distal-less in the posterior compartment. Scale bars, 50 μm. b’, Distal-less immunoreactivity was quantified in the indicated boxed regions and plotted separately for the anterior and posterior, where the glypicans were overexpressed. c, Dlp conditioned medium stabilizes GFP–Wingless in solution. Conditioned medium from S2 cells or S2 cells expressing DlpΔGPI–V5 was added to S2 cells expressing GFP–Wingless. Twelve hours later, the medium was collected, concentrated 20-fold and the amount of GFP–Wingless in solution was determined via western blot. d, Wingless solubilized by DlpΔGPI is signalling competent. Medium from cells that were mock-transfected, transfected with GFP–Wingless alone, or co-transfected with either V5 tagged DallyΔGPI or DlpΔGPI was collected, concentrated and added to S2R+ cells expressing a luciferase reporter of Wingless signalling. Error bars show standard deviation from the mean. n = 4, in which n denotes independent experiments and each independent experiment was performed in triplicate. Asterisk denotes statistical significance, as assessed by an unpaired, two-tailed t-test (P = 0.0011). ns, not significant (P = 0.128). All experiments were repeated independently three times with similar results.

Source data

Extended Data Fig. 6 Steric clashes prevent Wnt lipid binding to glypican CRD.

a, b, CRD architecture for Dlpcore in complex with palmitoleoylated serine of Wnt peptide. We could confirm that the DlpCRD contains the full set of canonical disulfide bonds. cf, Comparison of the conserved CRD architecture of apo Drosophila Dlpcore (PDB code 3odn) (c), human GPC1 (PDB code 4YWT) (d), mouse Frizzled 8 in complex with palmitoleic moiety from Xenopus Wnt8 (PDB code 4F0A) (e) and mouse Frizzled 8 in complex with palmitoleic moiety from human Wnt3 (PDB code 6AHY) (f). Evolutionary conserved disulfides are shown in black and numbered. g, h, Superposition of Dlpcore CRD and mouse Frizzled 8 CRD bound to lipidated Wnt8 (g) or Wnt3 (h), showing that a conserved helix of glypican CRD sterically hinders binding of the Wnt palmitoleate.

Extended Data Fig. 7 Additional structural information on Dlpcore in complex with human WNT7A peptide.

a, Sequence of Dlpcore construct from the complexed structure annotated with secondary structure elements. To facilitate comparison between the complexed and apo structure, secondary structure nomenclature of the complex reflects that of the previously published apo structure (PDB code 3odn). α indicates α-helices, η indicates 310-helices. b, Side and front view of the lipid binding cavity of Dlpcore in complex with the Wnt palmitoleoylated peptide, showing the cavity extension beyond the end of the Wnt peptide acyl chain. This additional space likely accommodates the bodipy moiety of bodipy–palmitate for the assays presented in Fig. 4a. The internal volume of the cavity is coloured in blue, with the palmitoleoylated serine from the Wnt peptide represented as spheres in atomic colouring (C: orange, N: blue, O: red).

Extended Data Fig. 8 Several types of omit maps show and support the modelling of the electron density in the binding pocket as PMS from the palmitoleoylated Wnt peptide.

a, Coot-displayed Fo − Fc omit map (left) generated from the refined model after removal of PMS, as shown in Fig. 3c. To help orientation and comparison, Fig. 3c is duplicated here, displaying the same map in Pymol (right). The maps are contoured at the same level in the two programs (±2.5σ). b, Coot-displayed Fo − Fc omit map (contoured at ±2.5σ) generated from the refined model, after removal of PMS, application of simulated annealing and one round of coordinate and B-factor refinement. c, Coot-displayed Fo − Fc map (left) and 2Fo − Fc map (right) of the last refinement iteration before modelling PMS in the electron density. As PMS was never modelled, these maps are unbiased. The Fo − Fc map and 2Fo − Fc map are contoured at ±2.5σ and 1σ, respectively. d, Polder map generated from the refined model using phenix.polder54. This omit map, the characteristic of which is to enhance weak electron density features in the omit region, shows additional electron density that can be assigned to the peptide portion of the palmitoleoylated peptide, therefore further supporting PMS modelling into the electron density. The map is contoured at ± 2.5σ.

Extended Data Fig. 9 In vivo characterization of Dlp/Dally chimaeras.

a, Multiple sequence alignment of residues forming binding pocket of human and D. melanogaster glypicans. Sequences are coloured according to Clustal X colouring scheme (blue, hydrophobics; green, polar). b, Extracellular distribution after overexpression of haemagglutinin-tagged Dlp, Dlp(F172T/F195Y; FF/TY), Dlp(R169E/T170N/Q171M/F172T; RTQF/ENMT) or Dally(E149R/N150T/M151Q/T152F; ENMT/RTQF) with ptc-gal4. Scale bars, 50 μm. c, Model illustrating how Dlp acts both as a reservoir of signalling competent Wnt and as a co-receptor. The weight of these activities depends on the relative abundance of Dlp to Frizzled and on the relative affinities of Dlp and Frizzled for Wnt. Configuration of glypican core and heparan sulfate chains are inspired from the structure of human GPC126. All molecules are drawn approximately to scale. The heparan sulfate chains are presented as orange circles, the glypican stalk regions from which they originate as black lines, and the GPI anchors as orange triangles.

Extended Data Table 1 Data collection and refinement statistics (molecular replacement)

Supplementary information

41586_2020_2498_MOESM1_ESM.pdf

Supplementary Information Supplementary Figure 1: Uncropped Western blot gels and molecular size marker. a) Blots correspond to the data from Figure 2d. b) Blots correspond to the data from Figure 2e. c-c’) Blots correspond to the data from Figure 4c. d) Blots correspond to the data from Extended Data 4b. e) Blots correspond to the data from Extended Data 5c. Supplementary Table 1: Table of Drosophila genotypes from this study.

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McGough, I.J., Vecchia, L., Bishop, B. et al. Glypicans shield the Wnt lipid moiety to enable signalling at a distance. Nature 585, 85–90 (2020). https://doi.org/10.1038/s41586-020-2498-z

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