Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure and mechanism of lysosome transmembrane acetylation by HGSNAT

Nature Structural & Molecular Biology (2024)Cite this article

Subjects

Abstract

Lysosomal transmembrane acetylation of heparan sulfates (HS) is catalyzed by HS acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), whose dysfunction leads to lysosomal storage diseases. The mechanism by which HGSNAT, the sole non-hydrolase enzyme in HS degradation, brings cytosolic acetyl-coenzyme A (Ac-CoA) and lysosomal HS together for N-acyltransferase reactions remains unclear. Here, we present cryogenic-electron microscopy structures of HGSNAT alone, complexed with Ac-CoA and with acetylated products. These structures explain that Ac-CoA binding from the cytosolic side causes dimeric HGSNAT to form a transmembrane tunnel. Within this tunnel, catalytic histidine and asparagine approach the lumen and instigate the transfer of the acetyl group from Ac-CoA to the glucosamine group of HS. Our study unveils a transmembrane acetylation mechanism that may help advance therapeutic strategies targeting lysosomal storage diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Functional characterization and architecture of human HGSNAT.
Fig. 2: Overall structure of the Ac-CoA substrate complex.
Fig. 3: Conformational changes between the apo and Ac-CoA-bound states.
Fig. 4: Overall structure of HGSNAT complexed with CoA–NAG or CoA–4MU-NAG.
Fig. 5: Proposed catalytic mechanism of human HGSNAT.

Similar content being viewed by others

Data availability

The sequence of human HGSNAT is available at the following link: https://www.uniprot.org/uniprotkb/Q68CP4/entry#sequences. The cryo-EM maps and coordinates of apo-HGSNAT, Ac-CoA-HGSNAT, CoA/NAG-HGSNAT and CoA/4MU-NAG-HGSNAT complexes have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-36376, EMD-36386, EMD-36387 and EMD-37264 and in the Protein Data Bank (PDB) under accession codes 8JKV, 8JL1, 8JL3 and 8W4A, respectively. Source data are provided with this paper.

References

  1. Parish, C. R. The role of heparan sulphate in inflammation. Nat. Rev. Immunol. 6, 633–643 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Sasisekharan, R., Shriver, Z., Venkataraman, G. & Narayanasami, U. Roles of heparan-sulphate glycosaminoglycans in cancer. Nat. Rev. Cancer 2, 521–528 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Simon Davis, D. A. & Parish, C. R. Heparan sulfate: a ubiquitous glycosaminoglycan with multiple roles in immunity. Front Immunol. 4, 470 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Long, K. R. & Huttner, W. B. How the extracellular matrix shapes neural development. Open Biol. 9, 180216 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Freeman, C. & Hopwood, J. Lysosomal degradation of heparin and heparan sulphate. Adv. Exp. Med. Biol. 313, 121–134 (1992).

    Article  CAS  PubMed  Google Scholar 

  6. Griffin, L. S. & Gloster, T. M. The enzymatic degradation of heparan sulfate. Protein Pept. Lett. 24, 710–722 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Andrade, F., Aldamiz-Echevarria, L., Llarena, M. & Couce, M. L. Sanfilippo syndrome: overall review. Pediatr. Int. 57, 331–338 (2015).

    Article  PubMed  Google Scholar 

  8. Bame, K. J. & Rome, L. H. Acetyl coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism. J. Biol. Chem. 260, 11293–11299 (1985).

    Article  CAS  PubMed  Google Scholar 

  9. Bame, K. J. & Rome, L. H. Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue. J. Biol. Chem. 261, 10127–10132 (1986).

    Article  CAS  PubMed  Google Scholar 

  10. Pshezhetsky, A. V. Lysosomal storage of heparan sulfate causes mitochondrial defects, altered autophagy, and neuronal death in the mouse model of mucopolysaccharidosis III type C. Autophagy 12, 1059–1060 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Hrebicek, M. et al. Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Am. J. Hum. Genet. 79, 807–819 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Meikle, P. J., Whittle, A. M. & Hopwood, J. J. Human acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation. Biochem. J. 308, 327–333 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Fan, X. et al. Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C). Am. J. Hum. Genet. 79, 738–744 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fedele, A. O. Sanfilippo syndrome: causes, consequences, and treatments. Appl Clin. Genet. 8, 269–281 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Platt, F. M. Emptying the stores: lysosomal diseases and therapeutic strategies. Nat. Rev. Drug Discov. 17, 133–150 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. Martins, C. et al. Molecular characterization of a large group of mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease. Hum. Mutat. 40, 1084–1100 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Feldhammer, M., Durand, S. & Pshezhetsky, A. V. Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C. PLoS ONE 4, e7434 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Fedele, A. O. & Hopwood, J. J. Functional analysis of the HGSNAT gene in patients with mucopolysaccharidosis IIIC (Sanfilippo C Syndrome). Hum. Mutat. 31, E1574–E1586 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Pan, X. et al. Glucosamine amends CNS pathology in mucopolysaccharidosis IIIC mouse expressing misfolded HGSNAT. J. Exp. Med. 219, e20211860 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Spahiu, L. et al. Mucopolysaccharidosis III: molecular basis and treatment. Pediatr. Endocrinol. Diabetes Metab. 27, 201–208 (2021).

    Article  PubMed  Google Scholar 

  21. Feldhammer, M. et al. Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT) gene. Hum. Mutat. 30, 918–925 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Durand, S., Feldhammer, M., Bonneil, E., Thibault, P. & Pshezhetsky, A. V. Analysis of the biogenesis of heparan sulfate acetyl-CoA:alpha-glucosaminide N-acetyltransferase provides insights into the mechanism underlying its complete deficiency in mucopolysaccharidosis IIIC. J. Biol. Chem. 285, 31233–31242 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Marmorstein, R. Structure and function of histone acetyltransferases. Cell. Mol. Life Sci. 58, 693–703 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Marmorstein, R. & Roth, S. Y. Histone acetyltransferases: function, structure, and catalysis. Curr. Opin. Genet Dev. 11, 155–161 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Salah Ud-Din, A. I., Tikhomirova, A. & Roujeinikova, A. Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). Int. J. Mol. Sci. https://doi.org/10.3390/ijms17071018 (2016).

  26. Christensen, D. G. et al. Post-translational protein acetylation: an elegant mechanism for bacteria to dynamically regulate metabolic functions. Front. Microbiol. 10, 1604 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Navratna, V., Kumar, A. & Mosalaganti, S. Structure of the human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT). eLife https://doi.org/10.7554/eLife.93510.1 (2024).

  28. Ma, D. et al. Crystal structure of a membrane-bound O-acyltransferase. Nature 562, 286–290 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Guan, C. et al. Structural insights into the inhibition mechanism of human sterol O-acyltransferase 1 by a competitive inhibitor. Nat. Commun. 11, 2478 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jiang, Y., Benz, T. L. & Long, S. B. Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT. Science 372, 1215–1219 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Liu, Y. et al. Mechanisms and inhibition of Porcupine-mediated Wnt acylation. Nature 607, 816–822 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Qian, H. et al. Structural basis for catalysis and substrate specificity of human ACAT1. Nature 581, 333–338 (2020).

    Article  CAS  PubMed  Google Scholar 

  33. Wang, L. et al. Structure and mechanism of human diacylglycerol O-acyltransferase 1. Nature 581, 329–332 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dutnall, R. N., Tafrov, S. T., Sternglanz, R. & Ramakrishnan, V. Structure of the histone acetyltransferase Hat1: a paradigm for the GCN5-related N-acetyltransferase superfamily. Cell 94, 427–438 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Chen, J. Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. Rep. 6, 31425 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liszczak, G., Arnesen, T. & Marmorstein, R. Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation. J. Biol. Chem. 286, 37002–37010 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wolf, E. et al. Crystal structure of a GCN5-related N-acetyltransferase: Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 94, 439–449 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Leaback, D. H. & Walker, P. G. Studies on glucosaminidase. 4. The fluorimetric assay of N-acetyl-beta-glucosaminidase. Biochem. J. 78, 151–156 (1961).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Voznyi, Ya,V. et al. A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C). J. Inherit. Metab. Dis. 16, 465–472 (1993).

    Article  CAS  Google Scholar 

  40. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, R. et al. Global stable-isotope tracing metabolomics reveals system-wide metabolic alternations in aging Drosophila. Nat. Commun. 13, 3518 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  45. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  47. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D. Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. The PyMOL Molecular Graphics System. v.2.3.5 (Schrodinger, 2020).

  50. Smart, O. S., Goodfellow, J. M. & Wallace, B. A. The pore dimensions of gramicidin A. Biophys. J. 65, 2455–2460 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Cryo-Electron Microscopy Center at the Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry for the help with data collection. We thank ShanghaiTech Cryo-EM Imaging Facility for its assistance in data collection. We thank the Molecular and Cell Biology Core Facility at the School of Life Science and Technology, ShanghaiTech University, for providing technical support. This work was supported by Shanghai Lingang Laboratory (grant nos. LG-QS-202203-03 to J.Y. and LG-QS-202203-05 to J.G.) and Shanghai Pujiang Program (grant no. 22PJ1410300 to J.G.).

Author information

Author notes

  1. These authors contributed equally: Ruisheng Xu, Yingjie Ning.

Authors and Affiliations

  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Ruisheng Xu, Fandong Ren, Zhengjiang Zhu & Jie Yu

  2. University of Chinese Academy of Sciences, Beijing, China

    Ruisheng Xu

  3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Yingjie Ning, Chenxia Gu & Jingpeng Ge

  4. Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine Research Centre, University of Montreal, Montreal, Quebec, Canada

    Xuefang Pan & Alexey V. Pshezhetsky

  5. Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

    Alexey V. Pshezhetsky

  6. Shanghai Key Laboratory of Aging Studies, Shanghai, China

    Jie Yu

Authors
  1. Ruisheng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yingjie Ning
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fandong Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chenxia Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhengjiang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xuefang Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexey V. Pshezhetsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jingpeng Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jie Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.Y. and J.G. designed the project. R.X. and Y.N. performed the sample preparation for cryo-EM. R.X., Y.N. and C.G. performed the biochemistry studies. R.X. performed the fluorescence-based acetylation assay. F.R. and Z.Z. carried out MS analysis. J.Y. and J.G. performed the cryo-EM data collection, data analysis and model building. J.Y., J.G., A.V.P. and X.P. wrote the manuscript.

Corresponding authors

Correspondence to Alexey V. Pshezhetsky, Jingpeng Ge or Jie Yu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Biochemical characterization and cryo-EM analysis of human HGSNAT.

a. Topology of HGSNAT. Unresolved regions are marked with dashed lines. b. Representative size exclusion chromatography (SEC) profile (left) of HGSNAT and SDS-PAGE gel (right) of HGSNAT in the presence or absence of β-ME. The trace and gel image are representative of 4 experimental replicates. c. A representative cryo-EM micrograph of HGSNAT. (out of 30,545 similar micrographs). d. Selected representative 2D class averages.

Source data

Extended Data Fig. 2 A representative flow chart of data processing and representative densities of the HGSNAT map.

a. Flow chart of data processing for Ac-CoA-bound HGSNAT complexes. Details may be found in Methods. b. Cryo-EM densities of the transmembrane helixes in the Ac-CoA-bound HGSNAT complexes.

Extended Data Fig. 3 3D reconstructions of the apo, Ac-CoA-bound, CoA/NAG-bound, and CoA/4MU-NAG-bound HGSNAT complexes.

a, e, j, o. Local resolution estimates of the apo (a), Ac-CoA-bound (e), CoA/NAG-bound (j), and CoA/4MU-NAG-bound (o) HGSNAT maps. b, f, k, p. FSC curves before and after masking and between the model and the final maps of the apo (b), Ac-CoA-bound (f), CoA/NAG-bound (k), and CoA/4MU-NAG-bound (p) HGSNAT. c, g, l, q. Projection orientation distribution of apo (c), Ac-CoA-bound (g), CoA/NAG-bound (l), and CoA/4MU-NAG-bound (q) HGSNAT maps, generated from cryoSPARC. d, h, m, r. The 3DFSC sphericity of the apo (d), Ac-CoA-bound (h), CoA/NAG-bound (m), and CoA/4MU-NAG-bound (r) HGSNAT maps, analyzed with 3DFSC in cryoSPARC. i, n, s. Densities of the substrates determined in the Ac-CoA-bound (i), CoA/NAG-bound (n), and CoA/4MU-NAG-bound (s) HGSNAT complexes. t. Densities of the Met282, Phe313, Lys341, and Arg345 in the apo and Ac-CoA-bound HGSNAT structures to illustrate the repositioning of these four residues upon the binding of Ac-CoA.

Source data

Extended Data Fig. 4 Effects of the indicated mutants on the activity of HGSNAT.

a. The relative enzyme activities of the indicated mutants. Data are shown as mean ± standard deviations, n = 3 biologically independent samples. b–s. Size exclusion chromatography profiles of the HGSNAT mutants.

Source data

Extended Data Fig. 5 Thermostability of wild type HGSNAT and HGSNAT mutants.

a–d. Melting curves of wild type HGSNAT, indicated HGSNAT mutants and CFP. The CFP tag, located at the C-terminal of the wild type HGSNAT and mutants, was used for protein detection. Data are shown as mean ± standard deviations, n = 3 biologically independent samples. e. The interactions in the NTD-TMD interface. Possible hydrogen bonds are indicated with dashed lines.

Source data

Extended Data Fig. 6 LC-MS identification of the endogenous Ac-CoA in HGSNAT.

a. LC profiles of commercial standard Ac-CoA (top) and purified HGSNAT enzyme from the solubilization of the whole cells without any additional dialysis (bottom). b. MS/MS spectrum of commercial standard Ac-CoA (top) and purified HGSNAT enzyme from the solubilization of the whole cells without any additional dialysis (bottom).

Source data

Extended Data Fig. 7 Sequence alignment of the HGSNAT across various species.

Secondary elements are labeled above the alignment. The catalytic residues Asn286 and His297 are highlighted and indicated with red stars.

Extended Data Fig. 8 The comparisons of the overall structure and the positions of catalytic site between HGSNAT with lipid-modifying MBOATs.

a–e. The overall structure of HGSNAT (a) in comparison with HHAT (b), Porcupine (c), ACAT1 (d), and DGAT1 (e). The acyl-CoA and hallmark catalytic histidine in each enzyme are indicated and shown in ball and stick style.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Video

Illustrating the transmembrane acetylation reaction catalyzed by HGSNAT.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, R., Ning, Y., Ren, F. et al. Structure and mechanism of lysosome transmembrane acetylation by HGSNAT. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01315-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41594-024-01315-5

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing