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Chemical editing of proteoglycan architecture

Nature Chemical Biology volume 18, pages 634–642 (2022)Cite this article

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Abstract

Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure–function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function.

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Fig. 1: Production of pPY-functionalized PG core protein and azido-GAGs to generate defined ectodomains.
Fig. 2: Binding of SDC1 to HSPG-binding proteins is influenced by GAG identity and multivalency.
Fig. 3: Membrane remodeling with engineered SDC1 ectodomains provides insight into regulation of mESC differentiation by PGs.
Fig. 4: Adhesion and spreading of mammary carcinoma cells.
Fig. 5: Proximity tagging with A-SDC145,47 to capture interactomes in live cells.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files. Please contact the corresponding author (M.H.) for access of raw data, which will be made available upon reasonable request. Source data are provided with this paper.

References

  1. Hacker, U., Nybakken, K. & Perrimon, N. Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530–541 (2005).

    Article  PubMed  CAS  Google Scholar 

  2. Critcher, M., O’Leary, T. & Huang, M. L. Glycoengineering: scratching the surface. Biochem. J. 478, 703–719 (2021).

    Article  CAS  PubMed  Google Scholar 

  3. Langford, J. K., Stanley, M. J., Cao, D. & Sanderson, R. D. Multiple heparan sulfate chains are required for optimal syndecan-1 function. J. Biol. Chem. 273, 29965–29971 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Bernfield, M. et al. Biology of the syndecans - a family of transmembrane heparan-sulfate proteoglycans. Annu Rev. Cell Biol. 8, 365–393 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. Zhang, Y. H. et al. Targeting of heparanase-modified Syndecan-1 by prosecretory mitogen lacritin requires conserved core GAGAL plus heparan and chondroitin sulfate as a novel hybrid binding site that enhances selectivity. J. Biol. Chem. 288, 12090–12101 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gama, C. I. et al. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat. Chem. Biol. 2, 467–473 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Manon-Jensen, T., Itoh, Y. & Couchman, J. R. Proteoglycans in health and disease: the multiple roles of syndecan shedding. FEBS J. 277, 3876–3889 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Salmivirta, M., Lidholt, K. & Lindahl, U. Heparan sulfate: a piece of information. FASEB J. 10, 1270–1279 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Nadanaka, S., Clement, A., Masayama, K., Faissner, A. & Sugahara, K. Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity. J. Biol. Chem. 273, 3296–3307 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Brittis, P. A., Canning, D. R. & Silver, J. Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science 255, 733–736 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Forsberg, E. & Kjellen, L. Heparan sulfate: lessons from knockout mice. J. Clin. Invest. 108, 175–180 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim, J. M., Lee, K., Kim, M. Y., Shin, H. I. & Jeong, D. Suppressive effect of syndecan ectodomains and N-desulfated heparins on osteoclastogenesis via direct binding to macrophage-colony stimulating factor. Cell Death Dis. https://doi.org/10.1038/s41419-018-1167-8 (2018).

  13. Schultz, K. C. et al. A genetically encoded infrared probe. J. Am. Chem. Soc. 128, 13984–13985 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Csizmar, C. M., Petersburg, J. R. & Wagner, C. R. Programming cell-cell interactions through non-genetic membrane engineering. Cell Chem. Biol. 25, 931–940 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Bundy, B. C. & Swartz, J. R. Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. Bioconjugate Chem. 21, 255–263 (2010).

    Article  CAS  Google Scholar 

  17. Gondelaud, F. et al. Extended disorder at the cell surface: the conformational landscape of the ectodomains of syndecans. Matrix Biol. 12, 100081 (2021).

    Article  CAS  Google Scholar 

  18. Fritz, T. A. & Esko, J. D. Xyloside priming of glycosaminoglycan biosynthesis and inhibition of proteoglycan assembly. Methods Mol. Biol. 171, 317–323 (2001).

    CAS  PubMed  Google Scholar 

  19. Dahbi, S. et al. Synthesis of a library of variously modified 4-methylumbelliferyl xylosides and a structure-activity study of human beta 4GalT7. Org. Biomol. Chem. 15, 9653–9669 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Carnachan, S. M. & Hinkley, S. F. R. Heparan sulfate identification and characterisation: method II. enzymatic depolymerisation and SAX-HPLC analysis to determine disaccharide composition. Bio-Protocol https://doi.org/10.21769/BioProtoc.2197 (2017).

  21. Trieger, G. W., Verespy, S., Gordts, P. L. S. M. & Godula, K. Efficient synthesis of heparinoid bioconjugates for tailoring FGF2 activity at the stem cell–matrix interface. Bioconjugate Chem. 30, 833–840 (2019).

    Article  CAS  Google Scholar 

  22. Burgess, R. R. A brief practical review of size exclusion chromatography: rules of thumb, limitations, and troubleshooting. Protein Expr. Purif. 150, 81–85 (2018).

    Article  CAS  PubMed  Google Scholar 

  23. Ibrahimi, O. A., Zhang, F., Hrstka, S. C. L., Mohammadi, M. & Linhardt, R. J. Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly. Biochemistry 43, 4724–4730 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Wilkins-Port, C. E., Sanderson, R. D., Tominna-Sebald, E. & McKeown-Longo, P. J. Vitronectin’s basic domain is a syndecan ligand which functions in trans to regulate vitronectin turnover. Cell Commun. Adhes. 10, 85–103 (2003).

    CAS  PubMed  Google Scholar 

  25. Beauvais, D. M., Burbach, B. J. & Rapraeger, A. C. The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells. J. Cell Biol. 167, 171–181 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Beauvais, D. M. & Rapraeger, A. C. Syndecan-1-mediated cell spreading requires signaling by αvβ3 integrins in human breast carcinoma cells. Exp. Cell. Res. 286, 219–232 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Powell, A. K., Fernig, D. G. & Turnbull, J. E. Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex. J. Biol. Chem. 277, 28554–28563 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Da Silva, M. S. et al. Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with α and β integrin subunits. Cell Commun. Adhes. 10, 59–67 (2009).

    Article  Google Scholar 

  29. Johnson, C. E. et al. Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells. Stem Cells 25, 1913–1923 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Boonyarattanakalin, S., Athavankar, S., Sun, Q. & Peterson, B. R. Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides. J. Am. Chem. Soc. 128, 386–387 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Woods, E. C., Yee, N. A., Shen, J. & Bertozzi, C. R. Glycocalyx engineering with a recycling glycopolymer that increases cell survival in vivo. Angew. Chem. Int. Ed. Engl. 54, 15782–15788 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hudak, A. et al. Contribution of syndecans to cellular uptake and fibrillation of alpha-synuclein and tau. Sci. Rep. 9, 16543 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Perez-Verdaguer, M. et al. Caveolin interaction governs Kv1.3 lipid raft targeting. Sci. Rep. 6, 22453 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chatterjee, S., Stephenson, T. N., Michalak, A. L., Godula, K. & Huang, M. L. Silencing glycosaminoglycan functions in mouse embryonic stem cells with small molecule antagonists. Method Enzymol. 626, 249–270 (2019).

    Article  CAS  Google Scholar 

  35. Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Vilen, Z., Joeh, E., Critcher, M., Parker, C. G. & Huang, M. L. Proximity tagging identifies the glycan-mediated glycoprotein interactors of galectin-1 in muscle stem cells. ACS Chem. Biol. https://doi.org/10.1021/acschembio.1c00313 (2021).

  37. Joeh, E. et al. Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling. Proc. Natl Acad. Sci. USA 117, 27329–27338 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Stepp, M. A., Pal-Ghosh, S., Tadvalkar, G. & Pajoohesh-Ganji, A. Syndecan-1 and its expanding list of contacts. Adv. Wound Care 4, 235–249 (2015).

    Article  Google Scholar 

  39. Tully, S. E. et al. A chondroitin sulfate small molecule that stimulates neuronal growth. J. Am. Chem. Soc. 126, 7736–7737 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Noti, C., de Paz, J. L., Polito, L. & Seeberger, P. H. Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions. Chem.-Eur. J. 12, 8664–8686 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Wang, Z. et al. Preactivation-based, one-pot combinatorial synthesis of heparin-like hexasaccharides for the analysis of heparin-protein interactions. Chem.-Eur. J. 16, 8365–8375 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Huang, M. L., Smith, R. A. A., Trieger, G. W. & Godula, K. Glycocalyx remodeling with proteoglycan mimetics promotes neural specification in embryonic stem cells. J. Am. Chem. Soc. 136, 10565–10568 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pulsipher, A., Griffin, M. E., Stone, S. E., Brown, J. M. & Hsieh-Wilson, L. C. Directing neuronal signaling through cell-surface glycan engineering. J. Am. Chem. Soc. 136, 6794–6797 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, W. Z. et al. Chemical synthesis of human syndecan-4 glycopeptide bearing O-, N-sulfation and multiple aspartic acids for probing impacts of the glycan chain and the core peptide on biological functions. Chem. Sci. 11, 6393–6404 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Qiu, H. et al. A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships. Nat. Methods 15, 889 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chen, Y. H. et al. The GAGOme: a cell-based library of displayed glycosaminoglycans. Nat. Methods 15, 881–888 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Nguyen, T. A., Cigler, M. & Lang, K. Expanding the genetic code to study protein-protein interactions. Angew. Chem. Int. Ed. 57, 14350–14361 (2018).

    Article  CAS  Google Scholar 

  49. Chatterjee, A., Sun, S. B., Furman, J. L., Xiao, H. & Schultz, P. G. A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52, 1828–1837 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Eriksson, A. S. & Spillmann, D. The mutual impact of Syndecan-1 and its glycosaminoglycan chains-a multivariable puzzle. J. Histochem. Cytochem. 60, 936–942 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Aquino, R. S., Teng, Y. H. & Park, P. W. Glycobiology of syndecan-1 in bacterial infections. Biochem. Soc. Trans. 46, 371–377 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Park, P. W., Pier, G. B., Hinkes, M. T. & Bernfield, M. Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411, 98–102 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Beauvais, D. M., Ell, B. J., McWhorter, A. R. & Rapraeger, A. C. Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J. Exp. Med. 206, 691–705 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gao, J., Xu, Y., Liu, J. & Huang, X. Convergent chemoenzymatic synthesis and biological evaluation of a heparan sulfate proteoglycan syndecan-1 mimetic. Chem. Commun. 57, 3407–3410 (2021).

    Article  CAS  Google Scholar 

  55. Zhang, P. et al. Heparan sulfate organizes neuronal synapses through neurexin partnerships. Cell 174, 1450 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Noborn, F. et al. Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans. Mol. Cell Proteom. 14, 41–49 (2015).

    Article  CAS  Google Scholar 

  57. Wiedemann, C., Bellstedt, P. & Gorlach, M. CAPITO—a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics 29, 1750–1757 (2013).

    Article  CAS  PubMed  Google Scholar 

  58. Siegbahn, A. et al. Exploration of the active site of β4GalT7: modifications of the aglycon of aromatic xylosides. Org. Biomol. Chem. 13, 3351–3362 (2015).

    Article  CAS  PubMed  Google Scholar 

  59. Trott, O. & Olson, A. J. Software news and update autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Yang, J. et al. Anti-beta2-microglobulin monoclonal antibodies induce apoptosis in myeloma cells by recruiting MHC class I to and excluding growth and survival cytokine receptors from lipid rafts. Blood 110, 3028–3035 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Chatterjee for assistance in preparing compound 2. We thank F. S. Ahmed and J. A. Hammond of the Scripps Research Biophysics and Biochemistry Core and D. S. Kojetin for assistance with circular dichroism experiments. For this work, T.N.S. (HD090292-S1), M.C. and M.L.H. are supported by the NIH K99/R00 Pathway to Independence Award (R00-HD090292) and NIGMS (R35GM142462). The acquisition of 600 MHz nuclear magnetic resonance spectra was supported by the NIH (S10OD021550). M.L.H. is grateful for the support and scientific counsel of K. Godula and J. Esko for this work.

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Author notes

  1. These authors contributed equally: Timothy R. O’Leary, Meg Critcher.

Authors and Affiliations

  1. Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA

    Timothy R. O’Leary, Meg Critcher, Tesia N. Stephenson, Xueyi Yang, Abdullah A. Hassan, Noah M. Bartfield, Richard Hawkins & Mia L. Huang

  2. Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA, USA

    Meg Critcher, Xueyi Yang & Mia L. Huang

  3. Department of Molecular Medicine, Scripps Research, La Jolla, CA, USA

    Mia L. Huang

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Contributions

T.R.O., M.C., T.N.S. and M.L.H. conceived and designed the research. T.R.O. performed molecular docking simulations. T.R.O. and X.Y. expressed engineered PGs and performed bioconjugation reactions. T.R.O. performed WAX and SEC characterization of ectodomains and M.C. performed circular dichroism experiments. T.R.O. and M.C. performed binding assays. T.N.S., N.M.B. and R.H. performed xyloside priming experiments and T.R.O., T.N.S. and N.M.B. performed GAG analysis. M.C. performed mESC differentiation, RT–qPCR, cell spreading assays and proximity tagging experiments. X.Y. contributed to biological assays. A.A.H. contributed the large-scale preparation of xylosides derivatives. T.R.O., M.C., T.N.S. and M.L.H. contributed to manuscript preparation.

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Correspondence to Mia L. Huang.

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Extended data

Extended Data Fig. 1 Characterization of engineered proteoglycan proteins.

(A) SDS-PAGE analysis and subsequent Coomassie (top) and fluorescence (bottom) imaging of SDC1, SDC2, SDC3 and SDC4 ectodomains reacted with TMR-azide confirms successful incorporation of pPY. As highly disordered proteins, SDCs run anomalously by SDS-PAGE. Representative of two technical replicates, molecular weight ladder in kDa. (B) Western blot analyses of SDC1 ectodomains probed with anti-SDC1 clone 281-2. (C) Intact mass spectrometry of SDC ectodomains confirms expected masses and demonstrates fidelity of pPY incorporation. Representative of two technical replicates, molecular weight ladder in kDa. N-terminal MQ residues were missing in some constructs, consistent with production and processing by E. coli. (D) Fluorescence microscopy images of beads following treatment with proteases or buffer with p-aminophenylmercuric acetate (PAPMA, required to activate MMP) shows SDC137TMR is only cleaved by MMP9 and trypsin (positive control). The TMR fluorophore is released from the beads after being washed, as it does not contain a poly-His tag sequence that enables the C-terminal fragment to remain anchored onto the nickel bead. (E) Circular dichroism (CD) spectrum of SDC1 ectodomains show >57% disordered conformations. APEX2 fusion proteins (A-SDC145,47 and A-SDC137,45,47) display enhanced helical structure relative to their non-fused counterparts due to the helical APEX220 domain. Ten readings were taken per sample to create an average CD spectrum. (F) Quantification of predicted secondary structures, generated by CAPITO analysis57.

Source data

Extended Data Fig. 2 Characterization of recombinant and azido-GAG.

(A) Graphical representation of heparan sulfate (HS) glycosaminoglycan biosynthesis in the endoplasmic reticulum (ER) and Golgi apparatus. The common HS and CS tetrasaccharide linker (Xyl-Gal-Gal-GlcA) is synthesized in the ER before being elongated by Ext1/2 enzyme complexes to create the HS repeating disaccharide motif. (B) Synthetic strategy to produce azidoxyloside 2. (C) Xyloside 2 was docked (AutoDock Vina) into the active site of β4GalT7 in complex with the UDP-Gal donor (teal, PDB ID 4M4K). Top poses for the docked compounds showed positioning of the xyloside C4 oxygen within 3-4 Å of the C1 atom on the galactose ring of UDP-galactose. The binding affinity of the five lowest energy poses for each simulation was averaged. Comparison of the average binding energies showed the addition of azido group into the aromatic ring component did not exact a substantial binding penalty. (D) Suspension CHO (CHO-S) cells are incubated with 2 (400 μM; 72 hr) and conditioned media is harvested for isolation of soluble recombinant, azido-GAGs (azGAGs). (E) SDS-PAGE analysis using AF546-appended rGAGs confirms that azide-primed GAGs are present mostly in the conditioned medium (CM) compared to cell extracts (E). Selective degradation of rGAGs by chondroitinase (Csase) results in a collapsed signal, indicative of our CM consisting primarily of CS. Representative of two biological replicates, molecular weight ladder in kDa. (F) azGAGs were analyzed by SDS-PAGE after copper-catalyzed azide-alkyne cycloaddition (CuAAC) with AF546 fluorophore and digestion with chondroitinase (CSase) or heparinase (HSase). The presence of a collapsed lower molecular-weight band upon CSase digestion suggests large amounts of primed CS. Representative of two biological replicates, molecular weight ladder in kDa. (G) Through dibenzocyclooctyne (DBCO) bead capture and subsequent disaccharide analysis, the proportion of azide-functionalized HS (orange) and CS (green) in the rGAG mixture can be quantified, with 27% being azido-HS (azHS), which mimics endogenous GAG ratios. (H) Standard curve of HS disaccharides. (I) Disaccharide analysis of endogenous HS (grey) from untreated cells, soluble non-primed HS (blue), azHS (orange) show similar sulfation profiles. Similarly, functionalization of free heparin (HEP, green) to azido-primed heparin (azHEP, yellow) did not affect sulfation. As expected, heparin is substantially more sulfated than HS. HS chains for mSDC1 (pink) were also analyzed. (J) Standard curve of CS disaccharides. (K) Disaccharide analysis of CS from endogenous (grey), azCS (green), rCS (blue) and mSDC1 (pink). (L) Proportion of CS and HS GAGs isolated from commercial, mammalian expressed mSDC1, calculated by disaccharide analysis. Analyses and graphs generated with GraphPad Prism 9. Bar graphs represent means and error bars represent SEM representative from two technical replicates.

Source data

Extended Data Fig. 3 Glycoconjugation of pPY-modified proteins by CuAAC.

(A) Weak anion exchange (WAX) traces demonstrating successful conjugation of azHEP to form SDC241,55,57HEP (mint), SDC380,82,89HEP (pink) and SDC444,62,64HEP (dark purple). SDC444,62,64 core protein (light purple) included as reference point for SDC core proteins. (B) Intact mass spectrometry of pPY-modified GFP (GFPyne(His)6), used as a model protein, confirms the appropriate mass shift (+630.4 Da) when reacted with tetramethylrhodamine (TMR)-azide. (C) WAX traces after reaction of GFPyne(His)6 with azHEP demonstrates a shift to the more anionic product (dark green). When the reaction is performed without copper (-Cu, red), the trace overlaps with WT GFP (grey). (D) WAX traces after reaction with azGAG (green) demonstrate a less anionic glycoconjugate than GFPyne + azHEP (dark green). Copper-free controls overlap with WT GFP peak. (E) SDS-PAGE analysis of WT and GFPyne(His)6 (Y) confirms the incorporation of pPY by TMR detection only in Y. azHEP conjugation results in a higher molecular weight smear, only in the presence of copper. Representative of two technical replicates, molecular weight ladder indicates kDa. (F) Circular dichroism spectra of heparin (red), SDC137 (grey), SDC137HEP (orange) and HEK239T expressed mSDC1 (pink). (G) Analysis of the predicted structures from CD spectra indicates that glycosylation of SDC137 does not impart a change, or increase, in protein structures and native, glycosylated SDC1 is highly disordered.

Source data

Extended Data Fig. 4 Additional AlphaScreen and ELISA binding data, including SDC380,82,89 and SDC444,62,64 glycoconjugates.

(A) Left: Graphical depiction of AlphaScreen assay using biotinylated interactors (FGF2, FGFR, vitronectin (VN)) attached to streptavidin donor beads and Ni-NTA acceptor beads which bind His-tagged SDC proteins and present them uniformly. Right: Graphical depiction of ELISA performed with randomly oriented immobilized SDC1 and recombinant integrin αvβ3. (B) AlphaScreens performed with biotinylated FGF2. (C) AlphaScreen performed with biotinylated FGFR1. (D) AlphaScreen performed with biotinylated FGFR1 with the addition of non-tagged FGF2 to stimulate ternary complex formation. (E) AlphaScreen performed with His-tagged SDC1 ectodomains and biotinylated vitronectin (VN). (F) ELISA performed with immobilized SDC1 and recombinant integrin αvβ3. Curves represent core proteins (filled, dotted), heparin conjugates (filled, dashed) and HS conjugates (open, dashed) of SDC380,82,89 (purple) and SDC444,62,64 (pink). (G) Bar graphs of EC50 from trivalent heparin (solid) and HS (dashed) SDC137,45,47, −380,82,89 and −444,62,64 glycoconjugates. Graphs were fitted using non-linear regression plotted using GraphPad Prism 9. Bar graphs represent means and error bars represent SEM generated from two technical replicates. (H) Tabulated apparent affinity constants (EC50) shown for SDC1 core proteins, SDC3 and SDC4 constructs, and heparan sulfate proteoglycan (HSPG)-binding proteins; FGF2, FGFR vitronectin (VN) and integrin αvβ3. mSDC1 represents HEK239T expressed recombinant mouse SDC1 ectodomain. EC50 tabulated at 1 significant figure.

Source data

Extended Data Fig. 5 Characterization of mESC remodeling by cholPEGNTA and additional differentiation data.

(A) Flow cytometry gating of Ext1−/− mESCs incubated with 10 µM cholPEGNTA for single cells (left), and GFP-negative population (right). (B) Treatment of Ext1−/− mESCs with 10 µM cholPEGNTA alone (untreated), or further incubation with varying concentrations of GFP(His)6 (3, 10 and 20 µM) demonstrates dose-dependent fluorescence. Comparisons of geometric mean GFP fluorescence from cells treated with 10 µM cholPEGNTA to GFP calibration beads allows for quantification of molecules of equivalent soluble fluorochrome (MESF) of protein on each cell. Saturation is observed at 10 µM GFP(His)6 at ~650,000 MESF. (C) Representative microscopy image of Ext1−/− cells treated with 10 µM cholPEGNTA for 1 hr and fixed at the indicated time points (hours after end of cholPEGNTA treatment) with 4% PFA/PBS. Cells were incubated with 10 µM GFP(His)6 and 100 μM Ni(OAc)2 for 1 hr in PBS before Hoechst staining. NTA headgroup remains accessible to His-tagged proteins for at least 8 hr. Data representative of two biological replicates. (D) Cartoon representation of Ext1−/− mESC treatment and ultracentrifugation for isolation of lipid rafts/caveolae in membrane fractions (green). (E) Dot blots from cholPEGNTA and GFP(His)6 treated Ext1−/− mESCs demonstrates striking overlap of CAV-1 (top) and SDC1 (middle) in lipid rafts. GFP(His)6 (bottom) was detected by fluorescence plate reader and quantified as a percentage of cholPEGNTA in each fraction (right). (F) Representative fluorescence microscopy images of mESCs on D6 of neuronal differentiation. Untreated Ext1−/− cells retain high Nanog expression, indicative of a pluripotent state, whilst mESCs differentiated with SDC1 constructs or soluble heparin lose Nanog expression (green). (G) RT–qPCR analysis of differentiated cells demonstrates decreased Nanog expression compared to untreated. Cells remodeled with cholPEGNTA for cell surface display of SDC1 proteins had lesser Nanog expression. (H) RT-qPCR analysis demonstrates significantly decreased expression of pluripotency marker Nanog at D6 upon treatment with heparin or SDC1 constructs. (I) Similar results to SDC1 are observed for Nanog expression when cells are treated with SDC3 and SDC4 proteins. (J) RT-qPCR analysis at D6 shows increased SOX1 expression when cells are treated with SDC3 and SDC4 proteins, both deglycosylated and as glycoconjugates. SDC4 shows significant differences between core protein (light purple) and its azHEP conjugate (purple), and between azHEP and azHS conjugates (dark purple). All experiments performed in technical triplicate in two biological replicates. One-sided ANOVA with Tukey’s post-hoc, p values indicated on graph, (****) P < 0.0001. Bar graphs represent means and error bars represent SEM.

Source data

Extended Data Fig. 6 Characterization of SDC1 knockdown by RNAi and CRISPR (SDC1KD) and additional cell spreading experiments.

(A) Representative microscopy images from soluble addition of monovalent SDC1 to wild-type MDA-MB-231. Data representative of three biological replicates. (B) Flow cytometry gating of wild-type MDA-MB-231 cells. (C) MDA-MB-231 cells treated with 100 nM (blue) or 200 nM (red) pooled SDC1 dsRNAi exhibit reduced SDC1 expression compared to non-targeting DsiRNA control (orange). Unstained cells (gray) are those incubated with secondary antibody only. (D) Quantification of SDC1 expression in MDA-MB-231 cells after knockdown with 100 nM and 200 nM pooled DsiRNA as a percent of non-targeting DsiRNA control. (E) Cartoon depiction of remodeling strategy of MDA-MB-231 cells, performed whilst cells are suspended in 96-well round bottom plates. Remodeled cells are plated on vitronectin-coated surfaces and allowed to spread for 2 hr. (F) Representative microscopy images of MDA-MB-231 cells treated with 200 nM hSDC1 siRNA or non-target. Only cell surface, glycosylated SDC1 proteins can rescue cell spreading. Data representative of three biological replicates. (G) Quantification of cell spreading on vitronectin. (H) Flow cytometry gating of wild-type MDA-MB-231 cells. (I) Quantification of SDC1 protein levels in CRISPR-generated SDC1KD cells by flow cytometry. Data represents the mean fluorescence intensity noramalised to WT MDA-MB-231 cells. (J) qRT-PCR confirms knockdown of SDC1 in SDC1KD cells using two primer sets targeting SDC1. Data presented is fold change of SDC1 mRNA as a percent of WT MDA-MB-231 cells, as calculated by delta delta CT. Bar graphs represent means and error bars represent SEM. One-sided ANOVA with Tukey’s post-hoc test with Šidák correction for multiple comparisons was performed; p values indicated on graph, (****) p < 0.0001. For each condition, n > 10 images examined across two biological replicates.

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Extended Data Fig. 7 APEX-SDC145,47 glycoconjugation yields and additional proximity labeling data.

(A) Weak anion exchange (WAX) traces demonstrating successful conjugation of azHEP to A-SDC145,47 (grey) and formation of the more anionic product A-SDC145,47HEP (blue). CuAAC performed at quantitative yields. (B) Dose dependent fluorescence generated after live cell proximity labeling with cell surface (10 µM cholPEGNTA) A-SDC145,47 at 5 µM, 2 µM and 1 µM. Biotinylation is detected by Cy5-streptavidin (pink) with fluorescence mostly localized to cell surfaces. Representative images from three biological replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Tables 1–5 and Notes 1 and 2.

Reporting Summary

Supplementary Data 1

Numerical source data for supplementary figures.

Supplementary Data 2

Unprocessed fluorescence gels and western blots for supplementary figures.

Source data

Source Data Fig. 1

Numerical source data for WAX and SEC plots.

Source Data Fig. 2

Numerical source data for AlphaScreen, ELISA and EC50 plots.

Source Data Fig. 3

Numerical source data for RT–qPCR plots.

Source Data Fig. 4

Numerical soured data for cell spreading plots.

Source Data Extended Data Fig. 1

Unprocessed gels and western blots.

Source Data Extended Data Fig. 1

Numerical source data for circular dichroism plots.

Source Data Extended Data Fig. 2

Unprocessed fluorescence gels.

Source Data Extended Data Fig. 2

Numerical source data for disaccharide composition plots.

Source Data Extended Data Fig. 3

Unprocessed fluorescence gels.

Source Data Extended Data Fig. 3

Numerical source data for WAX and circular dichroism plots.

Source Data Extended Data Fig. 4

Numerical source data for AlphaScreen, ELISA and EC50 plots.

Source Data Extended Data Fig. 5

Numerical source data for RT–qPCR plots.

Source Data Extended Data Fig. 6

Numerical source data for SDC1 KD validation.

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O’Leary, T.R., Critcher, M., Stephenson, T.N. et al. Chemical editing of proteoglycan architecture. Nat Chem Biol 18, 634–642 (2022). https://doi.org/10.1038/s41589-022-01023-5

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