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Primate-specific regulation of the human glycosphingolipid gatekeeper UGCG

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Data availability

The cryo-EM maps and atomic coordinates for the eight UGCG structures reported in this study have been deposited in the Electron Microscopy Data Bank and PDB, respectively, under the following accession codes: apo UGCG, EMD-80823 and PDB 26QF; UGCG bound to UDP-glucose, EMD-80832 and PDB 26QS; UGCG bound to UDP, EMD-80833 and PDB 26QT; UGCG bound to UDP-glucose and PS, EMD-80829 and PDB 26QM; UGCG bound to UDP and C6-ceramide, EMD-80824 and PDB 26QG; UGCG bound to miglustat, EMD-80827 and PDB 26QJ; UGCG bound to ibiglustat, EMD-80826 and PDB 26QI; and UGCG bound to eliglustat, EMD-80825 and PDB 26QH. The reference structure of Chlorella virus hyaluronan synthase used for structural comparison is available from the PDB under accession code 7SP7. Structural homology searches were performed against the PDB and AlphaFold Database. For molecular dynamics simulations, simulation input files, including initial system coordinate, topology and force-field files, equilibration configuration files and run scripts, as well as final output coordinate files, are available at Zenodo (https://doi.org/10.5281/zenodo.20809831)45. For sequence data sources: the amino acid sequences of UGCG analysed in this study were obtained from the NCBI protein database. The specific accession numbers for the species used in the phylogenetic analysis are as follows: Prototheria: Ornithorhynchus anatinus (platypus, XP_028909707.1), Tachyglossus aculeatus (echidna, XP_038626167.1); Metatheria: Monodelphis domestica (opossum, XP_001365801.1), Notamacropus eugenii (tammar wallaby, XP_072454167.1), Phascolarctos cinereus (koala, XP_020828114.1), Vombatus ursinus (wombat, XP_027695337.1), Sarcophilus harrisii (Tasmanian devil, XP_003761468.2); Afrotheria: Loxodonta africana (African elephant, XP_003407853.1), Trichechus manatus (manatee, XP_004372220.1), Orycteropus afer (aardvark, XP_007934906.1), Echinops telfairi (tenrec, XP_012862260.1); Xenarthra: Dasypus novemcinctus (armadillo, XP_058158524.1); Laurasiatheria: Erinaceus europaeus (hedgehog, XP_007529571.1), Myotis lucifugus (bat, XP_023609045.1), Equus caballus (Horse, XP_001490953.3), Tursiops truncatus (dolphin/whale, XP_073662506.1), Canis lupus familiaris (dog, NP_001165706.1), Felis catus (cat, XP_011286799.1), Manis javanica (pangolin, XP_017515964.1); Euarchontoglires: Mus musculus (mouse, NP_035803.1), Oryctolagus cuniculus (rabbit, XP_069911685.1), Tupaia belangeri (tree shrew, ELW70583.1), Carlito syrichta (tarsier, XP_008064016.1), Macaca mulatta (monkey, NP_001244652.1) and Homo sapiens (human, NP_003349.1). Source data are provided with this paper.

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Acknowledgements

The cryo-EM data were collected at the Shanghai Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Funding

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDB0830000 and XDB37030103 to H.E.X.); National Natural Science Foundation of China (grant nos. 825B2010 to J.J.W.; 32301016 to C.W.; 32130022 and 82495184 to H.E.X.; 82121005 to H.E.X. and Y.J.; and 82404881 to Q.Y.); The National Key R&D Program of China (grant no. 2022YFC2703105 to H.E.X.); National Key R&D Program ‘Strategic Scientific and Technological Innovation Cooperation’ Key Project (grant no. 2022YFE0203600) released by the Ministry of Science and Technology; Shanghai Municipal Science and Technology Major Project (grant no. 2019SHZDZX02 to H.E.X.); Program of Shanghai Academic/Technology Research Leader (grant no. 22XD1425200 to Y.J.) and the Shanghai Oriental Talents Program (Y.J.).

Author information

Author notes

  1. These authors contributed equally: Canrong Wu, Sanshan Jin

Authors and Affiliations

  1. Research Center for Medicinal Structural Biology, National Research Center for Translational Medicine at Shanghai, State Key Laboratory of Medical Genomics, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Canrong Wu, Jiuyin Xu, Changyao Li, Ming-Wei Wang & H. Eric Xu

  2. Lingang Laboratory, Shanghai, China

    Sanshan Jin, Mengting Jiang, Qingning Yuan & Yi Jiang

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

    Sanshan Jin, Mengting Jiang & Yi Jiang

  4. Division of Cardiology, Department of Internal Medicine and Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiologic Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    James Jiqi Wang

  5. School of Pharmacy, Nanjing Medical University, Nanjing, China

    Xiaoqi Guo

  6. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Fujian, China

    Yunhai Li

  7. Nanjing University of Chinese Medicine, Nanjing, China

    Zhenyu Cao & H. Eric Xu

  8. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Wen Hu, Youwei Xu & H. Eric Xu

  9. Research Center for Deepsea Bioresources, Sanya, China

    Ming-Wei Wang

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

    H. Eric Xu

Authors

  1. Canrong Wu
  2. Sanshan Jin
  3. Jiuyin Xu
  4. James Jiqi Wang
  5. Xiaoqi Guo
  6. Yunhai Li
  7. Zhenyu Cao
  8. Mengting Jiang
  9. Qingning Yuan
  10. Wen Hu
  11. Changyao Li
  12. Youwei Xu
  13. Ming-Wei Wang
  14. Yi Jiang
  15. H. Eric Xu

Contributions

S.J. and C.W. expressed and purified the proteins and prepared cryo-EM samples. Q.Y. and W.H. collected cryo-EM data. C.W. performed cryo-EM data processing and model building. C.W. conducted the enzyme assays. J.S. performed all molecular dynamics simulations and computational analyses. J.X., J.J.W., X.G., Y.L., Z.C., Y.X., C.L., M.J. and M.-W.W. provided experimental assistance and technical support. C.W., H.E.X., Y.J. and S.J. conceived of and designed the project. H.E.X., Y.J. and C.W. supervised the research and analysed the data. C.W. wrote the initial article draft and H.E.X. revised the paper with input from all authors. All authors reviewed and approved the final paper.

Corresponding authors

Correspondence to Canrong Wu, Yi Jiang or H. Eric Xu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Yu Cao, who co-reviewed with Kexin Hu; Binks W. Wattenberg, who co-reviewed with Usha Mahawar; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 Chain-length-dependent apparent competition and lateral-access model for ceramide engagement.

a, Apparent competition assay using unlabeled C6–C22 ceramides. Lower C6-NBD-glucosylceramide formation indicates stronger competition with the fluorescent acceptor substrate. C8-ceramide showed the strongest apparent competition, followed by C6-ceramide, whereas longer-chain ceramides showed weaker effects under the same delivery conditions. b, Docked/relaxed ceramide poses in the UGCG acceptor tunnel, comparing C6-, C8- and C16-ceramide. C16-ceramide was used as a representative long-chain ceramide. c, Membrane-coupled lateral-access model. The ceramide headgroup and proximal acyl segment enter the catalytic tunnel, whereas the distal acyl chain remains associated with the bilayer. Insets show representative docked/relaxed poses of C8- and C16-ceramide. Data in a are mean ± s.e.m.; statistical significance was assessed relative to DMSO. For statistical comparisons in a, n = 3 independent experiments. Significance thresholds are defined as follows: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 2 Functional and structural characterization of a non-productive phospholipid-occupied state.

a, Lipid inhibition screen of UGCG activity using 18:0–18:1 PC, PE, PG, PS and DAG under matched assay conditions. PS showed the strongest inhibition among the tested lipids, whereas PC and DAG had little effect. Ibiglustat was included as a positive inhibition control. Data are mean ± s.e.m.; statistical significance was assessed relative to DMSO. For statistical comparisons in a, n = 3 independent experiments. Significance thresholds are defined as follows: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data. b, Dose–response analysis of PS inhibition. The apparent IC50 for 18:0–18:1 PS was 112.2 ± 6.8 μM. c, Cryo-EM density and model of the UDP-glucose-bound, phospholipid-occupied UGCG state. The phospholipid-like density is located in the membrane-facing ceramide-access tunnel. d, Isolated density for the bound phospholipid, shown in two orientations, with the fitted PS model. e, Ligand RMSD of the fitted PS molecule from three independent 500-ns MD simulations, supporting the stability of the PS-bound configuration in the ceramide-access tunnel.

Source data

Extended Data Fig. 3 Ligand-responsive remodeling of the C-terminal β-hairpin.

a, Electrostatic surface representations of apo and UDP-glucose-bound UGCG, showing opening and occlusion of the cytosolic donor-binding pocket. b, Cartoon representations highlighting the C-terminal β-hairpin in the apo and UDP-glucose-bound states. The β-hairpin adopts a more compact conformation over the donor-binding pocket upon UDP-glucose binding. c, Structural superposition of apo and UDP-glucose-bound UGCG showing an approximately 5 Å displacement of the β-hairpin tip toward the donor-binding pocket. The hinge-proximal residue T372 is distal to UDP-glucose, indicating that the effect of the T372P mutation is not caused by loss of a direct ligand contact. d, UDP-glucose-dependent activity of wild-type UGCG and T372P. The T372P substitution increases the apparent Km for UDP-glucose and reduces apparent kcat, supporting a role for local β-hairpin flexibility in productive donor-pocket remodeling. Data are mean ± s.e.m. from independent experiments.

Source data

Extended Data Fig. 4 Molecular dynamics analysis of C-terminal β-hairpin remodeling.

a, Backbone RMSD of the C-terminal β-hairpin in apo and UDP-glucose-bound UGCG from three independent 500-ns MD simulations. Apo UGCG shows greater β-hairpin conformational variability, whereas the UDP-glucose-bound state maintains a more restrained β-hairpin conformation. b, Schematic showing the distance measurement used to compare β-hairpin positioning relative to the catalytic center. The distance was defined between the backbone centroid of the selected β-hairpin segment, residues Ile373–Tyr380, and the backbone atoms of Ser145. c, Violin plot showing the distribution of the β-hairpin centroid–S145 distance from the MD trajectories. UDP-glucose binding shifts the β-hairpin toward S145, consistent with a more compact, inward conformation over the donor-binding pocket. Dotted lines indicate the median and quartiles. Data are mean ± s.e.m. from n = 3 independent experiments.

Extended Data Fig. 5 Cellular and dynamic analysis of the Tyr196 modulatory checkpoint.

a, Cell-based C6-NBD-ceramide glucosylation assay in cells expressing human UGCG WT, human UGCG Y196S, mouse UGCG WT or mouse UGCG S196Y. Formation of C6-NBD-glucosylceramide was quantified and normalized to human UGCG WT. Human Y196S increased cellular glucosylation activity, whereas the reciprocal mouse S196Y substitution reduced activity relative to mouse WT, supporting a conserved activity-dampening effect of tyrosine at position 196. Data are mean ± s.e.m.; statistical significance was assessed relative to the corresponding WT enzyme. For statistical comparisons in a, n = 3 independent experiments. Significance thresholds are defined as follows: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data. b, Definition of the Tyr196 orientation angle relative to the membrane-normal Z axis. Representative apo and UDP/C6-ceramide-bound UGCG structures illustrate the upward/occluded and downward/open Tyr196 conformations, respectively. The inset shows local contacts associated with the two states, including the Tyr196–R280 contact in the upward/occluded conformation and the Tyr196–E295 contact in the downward/open conformation. c, Tyr196 angle distributions from three independent 500-ns MD simulations across eight UGCG states. Apo, UDP-bound, UDP-glucose-bound and miglustat-bound states predominantly sample the upward/occluded conformation, whereas UDP/C6-ceramide-bound, UDP-glucose/PS-occupied, eliglustat-bound and ibiglustat-bound states preferentially sample the downward/open conformation. Angles >90° correspond to upward/occluded orientations, and angles <90° correspond to downward/open orientations.

Source data

Extended Data Fig. 6 Functional validation of inhibitor-binding modes.

a, Dose–response curves for eliglustat inhibition of wild-type UGCG and selected eliglustat-pocket mutants, including Y196A and Y196S, retaining sufficient basal activity for IC50 determination. b, ΔpIC50 values for eliglustat-pocket mutants relative to wild type. ΔpIC50 was calculated as pIC50 (mutant) − pIC50 (WT); negative values indicate reduced inhibitor potency. Dashed lines indicate 10-fold and 100-fold decreases in apparent potency. c, Dose–response curves for ibiglustat inhibition of wild-type UGCG and selected ibiglustat-pocket mutants, including Y196A and Y196S. d, ΔpIC50 values for ibiglustat-pocket mutants relative to wild type, calculated as in b. e, Dose-response curves for miglustat inhibition of wild-type UGCG and M211A, an activity-retaining probe adjacent to the miglustat-binding environment. f, Eliglustat dose–response curves measured at low and high C6-NBD-ceramide concentrations. The rightward shift at higher C6-NBD-ceramide concentration is consistent with competition at or near the acceptor site. g, Miglustat dose–response curves measured at low and high UDP-glucose concentrations. The rightward shift at higher UDP-glucose concentration is consistent with competition at the donor-binding pocket. Data are mean ± s.e.m. For b and d, statistical significance was assessed relative to WT pIC50 values. For statistical comparisons in b and d, n = 3 independent experiments. Significance thresholds are defined as follows: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data. Y196A and Y196S had little effect on eliglustat potency but modestly reduced ibiglustat potency.

Source data

Extended Data Fig. 7 Working model for coordinated donor- and acceptor-site engagement by UGCG.

Schematic summary of UGCG substrate engagement, catalysis and inhibition at the Golgi membrane. The apo, UDP-glucose-bound and UDP/C6-ceramide-bound states correspond to experimentally observed structural states. UDP-glucose accesses the cytosolic donor pocket, whereas ceramide engages the membrane-facing acceptor tunnel through lateral access from the bilayer. Miglustat primarily blocks donor-pocket engagement, whereas eliglustat and ibiglustat block acceptor-tunnel engagement. The catalytic assembly and product-like state are shown as inferred models based on the observed structural snapshots. Tyr196 adopts an upward/occluded conformation in the apo and UDP-glucose-bound states and a downward/open conformation in the acceptor-engaged and inferred catalytic states. UDP and glucosylceramide release are depicted without assigning a release order. Enhanced dashed arrows indicate possible or modelled transitions; the order of substrate binding and product release remains unresolved.

Extended Data Table 1 Cryo-EM data collection, processing, refinement and validation statistics for apo, donor-bound, product-bound and phospholipid-occupied UGCG structures

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Extended Data Table 2 Cryo-EM data collection, processing, refinement and validation statistics for acceptor-bound and inhibitor-bound UGCG structures

Full size table

Supplementary information

Source data

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Wu, C., Jin, S., Xu, J. et al. Primate-specific regulation of the human glycosphingolipid gatekeeper UGCG. Nature (2026). https://doi.org/10.1038/s41586-026-10927-4

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