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Cited by 45Proteoglycans and glycosaminoglycans researchGlycosylation and Glycoproteins ResearchPolysaccharides and Plant Cell Walls

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Structure of the human heparan sulfate polymerase complex EXT1-EXT2

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Leisico, Francisco, Omeiri, Juneina, Le Narvor, Christine, Beaudouin, Joël, Hons, Michael, Fenel, Daphna, Schoehn, Guy, Couté, Yohann, Bonnaffé, David, Sadir, Rabia, Lortat-Jacob, Hugues, Wild, Rebekka·2022·Nature Communications
DOI 10.1038/s41467-022-34882-6PMC PMC9675754

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Abstract

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N -acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N -acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.

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Research works printed but not linked: 68

  1. Karamanou, K., Espinosa, D. C. R., Fortuna-Costa, A. & Pavão, M. S. G. Biological function of unique sulfated glycosaminoglycans in primitive chordates. Glycoconj. J. 34, 277–283 (2017). · DOI
  2. Yamada, S., Sugahara, K. & Özbek, S. Evolution of glycosaminoglycans. Commun. Integr. Biol. 4, 150–158 (2011). · DOI
  3. Couchman, J. R. & Pataki, C. A. An introduction to proteoglycans and their localization. J. Histochem. Cytochem 60, 885–897 (2012). · DOI
  4. Iozzo, R. V. & Schaefer, L. Proteoglycan form and function: A comprehensive nomenclature of proteoglycans. Matrix Biol. 42, 11–55 (2015). · DOI
  5. Li, J.-P. & Kusche-Gullberg, M. Heparan sulfate: biosynthesis, structure, and function. Int. Rev. Cell Mol. Biol. 325, 215–273 (2016). · DOI
  6. Annaval, T. et al. Heparan sulfate proteoglycans biosynthesis and post synthesis mechanisms combine few enzymes and few core proteins to generate extensive structural and functional diversity. Mol. Basel Switz. 25, E4215 (2020).
  7. Karamanos, N. K. et al. Proteoglycan chemical diversity drives multifunctional cell regulation and therapeutics. Chem. Rev. 118, 9152–9232 (2018). · DOI
  8. Moon, S. & Zhao, Y.-T. Spatial, temporal and cell-type-specific expression profiles of genes encoding heparan sulfate biosynthesis enzymes and proteoglycan core proteins. Glycobiology 31, 1308–1318 (2021). · DOI
  9. Clausen, T. M. et al. SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2. Cell 183, 1043–1057.e15 (2020). · DOI
  10. Gubbiotti, M. A., Buraschi, S., Kapoor, A. & Iozzo, R. V. Proteoglycan signaling in tumor angiogenesis and endothelial cell autophagy. Semin. Cancer Biol. 62, 1–8 (2020). · DOI
  11. Li, J.-P. & Zhang, X. Implications of heparan sulfate and heparanase in amyloid diseases. Adv. Exp. Med. Biol. 1221, 631–645 (2020). · DOI
  12. Tsiantoulas, D. et al. APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans. Nature 597, 92–96 (2021). · DOI
  13. Briggs, D. C. & Hohenester, E. Structural basis for the initiation of glycosaminoglycan biosynthesis by human Xylosyltransferase 1. Struct. Lond. Engl. 1993, 801–809.e3 (2018).
  14. Pedersen, L. C. et al. Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis. J. Biol. Chem. 278, 14420–14428 (2003). · DOI
  15. Pinhal, M. A. et al. Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo. Proc. Natl Acad. Sci. USA 98, 12984–12989 (2001). · DOI
  16. Bethea, H. N., Xu, D., Liu, J. & Pedersen, L. C. Redirecting the substrate specificity of heparan sulfate 2-O-sulfotransferase by structurally guided mutagenesis. Proc. Natl Acad. Sci. USA 105, 18724–18729 (2008). · DOI
  17. Pedersen, L. C. et al. Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I. J. Biol. Chem. 275, 34580–34585 (2000). · DOI
  18. Cheung, P. K. et al. Etiological point mutations in the hereditary multiple exostoses gene EXT1: a functional analysis of heparan sulfate polymerase activity. Am. J. Hum. Genet. 69, 55–66 (2001). · DOI
  19. Park, K. J. et al. Germline mutations in the EXT1 and EXT2 genes in Korean patients with hereditary multiple exostoses. J. Hum. Genet 44, 230–234 (1999). · DOI
  20. Pacifici, M. The pathogenic roles of heparan sulfate deficiency in hereditary multiple exostoses. Matrix Biol. J. Int. Soc. Matrix Biol. 71–72, 28–39 (2018). · DOI
  21. Busse, M. & Kusche-Gullberg, M. In vitro polymerization of heparan sulfate backbone by the EXT proteins. J. Biol. Chem. 278, 41333–41337 (2003). · DOI
  22. Senay, C. et al. The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis. EMBO Rep. 1, 282–286 (2000). · DOI
  23. Lind, T., Tufaro, F., McCormick, C., Lindahl, U. & Lidholt, K. The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem. 273, 26265–26268 (1998). · DOI
  24. McCormick, C., Duncan, G., Goutsos, K. T. & Tufaro, F. The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate. Proc. Natl Acad. Sci. USA 97, 668–673 (2000). · DOI
  25. Hassinen, A. & Kellokumpu, S. Organizational interplay of Golgi N-glycosyltransferases involves organelle microenvironment-dependent transitions between enzyme homo- and heteromers. J. Biol. Chem. 289, 26937–26948 (2014). · DOI
  26. Jackson, P. The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem. J. 270, 705–713 (1990). · DOI
  27. Klumperman, J. Architecture of the mammalian Golgi. Cold Spring Harb. Perspect. Biol. 3, a005181 (2011). · DOI
  28. Breton, C., Snajdrová, L., Jeanneau, C., Koca, J. & Imberty, A. Structures and mechanisms of glycosyltransferases. Glycobiology 16, 29R–37R (2006). · DOI
  29. Li, Z. et al. Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1). Nat. Commun. 8, 185 (2017). · DOI
  30. Holm, L. Using Dali for protein structure comparison. Methods Mol. Biol. Clifton NJ 2112, 29–42 (2020). · DOI
  31. Wuyts, W. et al. Identification and characterization of a novel member of the EXT gene family, EXTL2. Eur. J. Hum. Genet. EJHG 5, 382–389 (1997). · DOI
  32. Nadanaka, S. et al. EXTL2, a member of the EXT family of tumor suppressors, controls glycosaminoglycan biosynthesis in a xylose kinase-dependent manner. J. Biol. Chem. 288, 9321–9333 (2013). · DOI
  33. Wilson, L. F. L. et al. The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis. Nat. Commun. 13, 3314 (2022). · DOI
  34. Taujale, R. et al. Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases. eLife 9, e54532 (2020). · DOI
  35. Schuman, B., Evans, S. V. & Fyles, T. M. Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism. PLoS ONE 8, e71077 (2013). · DOI
  36. Okada, M., Nadanaka, S., Shoji, N., Tamura, J.-I. & Kitagawa, H. Biosynthesis of heparan sulfate in EXT1-deficient cells. Biochem. J. 428, 463–471 (2010). · DOI
  37. Bukowska-Olech, E. et al. Hereditary multiple exostoses-a review of the molecular background, diagnostics, and potential therapeutic strategies. Front. Genet. 12, 759129 (2021). · DOI
  38. Napiórkowska, M. et al. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nat. Struct. Mol. Biol. 24, 1100–1106 (2017). · DOI
  39. Wild, R. et al. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Science 359, 545–550 (2018). · DOI
  40. Schmid, J., Heider, D., Wendel, N. J., Sperl, N. & Sieber, V. Bacterial glycosyltransferases: challenges and opportunities of a highly diverse enzyme class toward tailoring natural products. Front. Microbiol. 7, 182 (2016). · DOI
  41. Moremen, K. W. & Haltiwanger, R. S. Emerging structural insights into glycosyltransferase-mediated synthesis of glycans. Nat. Chem. Biol. 15, 853–864 (2019). · DOI
  42. Yakovlieva, L. & Walvoort, M. T. C. Processivity in bacterial glycosyltransferases. ACS Chem. Biol. 15, 3–16 (2020). · DOI
  43. Hashimoto, K., Madej, T., Bryant, S. H. & Panchenko, A. R. Functional states of homooligomers: insights from the evolution of glycosyltransferases. J. Mol. Biol. 399, 196–206 (2010). · DOI
  44. Noffz, C., Keppler-Ross, S. & Dean, N. Hetero-oligomeric interactions between early glycosyltransferases of the dolichol cycle. Glycobiology 19, 472–478 (2009). · DOI
  45. De Pasquale, V., Quiccione, M. S., Tafuri, S., Avallone, L. & Pavone, L. M. Heparan sulfate proteoglycans in viral infection and treatment: a special focus on SARS-CoV-2. Int. J. Mol. Sci. 22, 6574 (2021). · DOI
  46. Lanzi, C. & Cassinelli, G. Heparan sulfate mimetics in cancer therapy: the challenge to define structural determinants and the relevance of targets for optimal activity. Molecules 23, 2915 (2018). · DOI
  47. Gedevanishvili, M. D. & Tsutsunava, L. E. [Pharmacologic analysis of the stimulating effect of serotonin on proliferation in cell cultures]. Tsitologiia 20, 686–689 (1978).
  48. Lucas, R., Hamza, D., Lubineau, A. & Bonnaffé, D. Synthesis of glycosaminoglycan oligosaccharides − an unexpected inhibitory effect of a remote N-Acetyl group upon trichloroacetimidate-mediated couplings. Eur. J. Org. Chem. 2004, 2107–2117 (2004). · DOI
  49. Hamza, D. et al. First synthesis of heparan sulfate tetrasaccharides containing both N-acetylated and N-unsubstituted glucosamine-search for putative 10E4 epitopes. ChemBioChem 7, 1856–1858 (2006). · DOI
  50. Povie, G. et al. Repairing the thiol-ene coupling reaction. Angew. Chem. Int. Ed. Engl. 53, 3894–3898 (2014). · DOI
  51. Kandiah, E. et al. CM01: a facility for cryo-electron microscopy at the European Synchrotron. Acta Crystallogr. Sect. Struct. Biol. 75, 528–535 (2019). · DOI
  52. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020). · DOI
  53. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM. Struct. Determ. J. Struct. Biol. 180, 519–530 (2012). · DOI · Paper page
  54. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). · DOI
  55. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). · DOI
  56. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007). · DOI
  57. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014). · DOI
  58. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). · DOI
  59. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010). · DOI · Paper page
  60. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. Struct. Biol. 74, 531–544 (2018). · DOI
  61. Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. Publ. Protein Soc. 27, 293–315 (2018). · DOI · Paper page
  62. Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. Publ. Protein Soc. 27, 14–25 (2018). · DOI · Paper page
  63. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014). · DOI · Paper page
  64. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016). · DOI · Paper page
  65. Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42, e168 (2014). · DOI · Paper page
  66. Laguri, C., Sadir, R., Gout, E., Vivès, R. R. & Lortat-Jacob, H. Preparation and characterization of heparan sulfate-derived oligosaccharides to investigate protein-GAG interaction and HS biosynthesis enzyme activity. Methods Mol. Biol. Clifton NJ 2303, 121–137 (2022). · DOI
  67. Casabona, M. G., Vandenbrouck, Y., Attree, I. & Couté, Y. Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane. Proteomics 13, 2419–2423 (2013). · DOI
  68. Bouyssié, D. et al. Proline: an efficient and user-friendly software suite for large-scale proteomics. Bioinforma. Oxf. Engl. 36, 3148–3155 (2020).

Metadata

Authors
Leisico, F., Omeiri, J., Le Narvor, C., Beaudouin, J., Hons, M., Fenel, D., Schoehn, G., Couté, Y., Bonnaffé, D., Sadir, R., Lortat-Jacob, H., & Wild, R.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • European Molecular Biology Laboratory (FR)
Corresponding author
Wild, Rebekka
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-11-19
Volume
13
Issue
1
Pages
7110-7110
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-34882-6
PMC
PMC9675754
OpenAlex
W4309562772
Cited by
45
References
3
Citations by year
2026: 16 · 2025: 9 · 2024: 10 · 2023: 10
FWCI
3.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • HAL (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Agence Nationale de la Recherche (ANR-10-INBS-08, ANR-18-CE11-0006-01, ANR-15-IDEX-02, ATIP-Avenir)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Proteoglycans and glycosaminoglycans research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Polysaccharides and Plant Cell Walls
Keywords
heparan sulfate biosynthesis, EXT1, EXT2, N-acetylglucosamine transferase, cryo-EM structure
MeSH terms
Heparitin Sulfate, Heparan Sulfate, Humans, Nucleotidyltransferases, Proteins, Glycosyltransferases, N-Acetylglucosaminyltransferases, Cryoelectron Microscopy

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