Skip to main content

Thank you for visiting 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.

Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3


Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3’s structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: In vitro evidence that GalNAc-T3 glycosylates FGF23 Thr178 using its lectin domain.
Fig. 2: Glycosylation of Thr178 in FGF23 by GalNAc-T3 in cells requires its lectin domain activity.
Fig. 3: Glycosylation kinetics of human GalNAc-T3 against a series of (glyco)peptides.
Fig. 4: Crystal structures of TgGalNAc-T3 in ternary complexes with UDP-Mn+2-P3 and UDP-Mn+2-FGF23c.
Fig. 5: Structural features of the peptide, UDP and lectin-domain GalNAc-binding sites of TgGalNAc-T3.
Fig. 6: Mapping HsGalNAc-T3 FTC mutations to the structure of TgGalNAc-T3.

Data availability

The crystal structures of TgGalNAc-T3-UDP-P3 and TgGalNAc-T3-UDP–FGF23c complexes were deposited at the RCSB PDB with accession codes 6S24 and 6S22, respectively.


  1. 1.

    Hurtado-Guerrero, R. Recent structural and mechanistic insights into protein O-GalNAc glycosylation. Biochem Soc. Trans. 44, 61–67 (2016).

    CAS  PubMed  Google Scholar 

  2. 2.

    Bennett, E. P. et al. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22, 736–756 (2012).

    CAS  PubMed  Google Scholar 

  3. 3.

    de Las Rivas, M., Lira-Navarrete, E., Gerken, T. A. & Hurtado-Guerrero, R. Polypeptide GalNAc-Ts: from redundancy to specificity. Curr. Opin. Struct. Biol. 56, 87–96 (2019).

    PubMed  Google Scholar 

  4. 4.

    Lira-Navarrete, E. et al. Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase 2. Angew. Chem. Int. Ed. Engl. 53, 8206–8210 (2014).

    CAS  PubMed  Google Scholar 

  5. 5.

    Gerken, T. A. et al. Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases. J. Biol. Chem. 286, 14493–14507 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Gerken, T. A. et al. The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation. J. Biol. Chem. 288, 19900–19914 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Revoredo, L. et al. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 26, 360–376 (2016).

    CAS  PubMed  Google Scholar 

  8. 8.

    Lira-Navarrete, E. et al. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat. Commun. 6, 6937 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Rivas, M. L. et al. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat. Commun. 8, 1959 (2017).

    PubMed Central  Google Scholar 

  10. 10.

    de Las Rivas, M. et al. Structural analysis of a GalNAc-T2 mutant reveals an induced-fit catalytic mechanism for GalNAc-Ts. Chemistry 24, 8382–8392 (2018).

    PubMed  Google Scholar 

  11. 11.

    Topaz, O. et al. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat. Genet. 36, 579–581 (2004).

    CAS  PubMed  Google Scholar 

  12. 12.

    Kato, K. et al. Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J. Biol. Chem. 281, 18370–18377 (2006).

    CAS  PubMed  Google Scholar 

  13. 13.

    White, K. E. et al. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat. Genet 26, 345–348 (2000).

    CAS  Google Scholar 

  14. 14.

    Benet-Pages, A., Orlik, P., Strom, T. M. & Lorenz-Depiereux, B. An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum. Mol. Genet. 14, 385–390 (2005).

    CAS  PubMed  Google Scholar 

  15. 15.

    Simpson, M. A. et al. Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia. Clin. Genet. 75, 271–276 (2009).

    CAS  PubMed  Google Scholar 

  16. 16.

    Tagliabracci, V. S. et al. Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis. Proc. Natl Acad. Sci. USA 111, 5520–5525 (2014).

    CAS  PubMed  Google Scholar 

  17. 17.

    Urakawa, I. et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444, 770–774 (2006).

    CAS  PubMed  Google Scholar 

  18. 18.

    Bennett, E. P., Hassan, H. & Clausen, H. cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-d-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3. J. Biol. Chem. 271, 17006–17012 (1996).

    CAS  PubMed  Google Scholar 

  19. 19.

    Kong, Y. et al. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis. Glycobiology 25, 55–65 (2015).

    CAS  PubMed  Google Scholar 

  20. 20.

    Schjoldager, K. T. et al. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome. EMBO Rep. 16, 1713–1722 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Khetarpal, S. A. et al. Loss of function of GALNT2 lowers high-density lipoproteins in humans, nonhuman primates, and rodents. Cell Metab. 24, 234–245 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Wang, S. et al. Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions. J. Biol. Chem. 293, 7408–7422 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Yoshimura, Y. et al. Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates. Glycobiology 22, 429–438 (2012).

    CAS  PubMed  Google Scholar 

  24. 24.

    Frishberg, Y. et al. Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J. Bone Min. Res 22, 235–242 (2007).

    CAS  Google Scholar 

  25. 25.

    Chen, G. et al. alpha-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling. Nature 553, 461–466 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Hagen, F. K., Hazes, B., Raffo, R., deSa, D. & Tabak, L. A. Structure-function analysis of the UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. J. Biol. Chem. 274, 6797–6803 (1999).

    CAS  PubMed  Google Scholar 

  27. 27.

    Kozarsky, K., Kingsley, D. & Krieger, M. Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors. Proc. Natl Acad. Sci. USA 85, 4335–4339 (1988).

    CAS  PubMed  Google Scholar 

  28. 28.

    Bar-Even, A. et al. The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50, 4402–4410 (2011).

    CAS  PubMed  Google Scholar 

  29. 29.

    de Las Rivas, M. et al. Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4. ACS Cent. Sci. 4, 1274–1290 (2018).

    PubMed  Google Scholar 

  30. 30.

    Lira-Navarrete, E. et al. Structural insights into the mechanism of protein O-fucosylation. PLoS ONE 6, e25365 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ghirardello, M. et al. Glycomimetics targeting glycosyltransferases: synthetic, computational and structural studies of less-polar conjugates. Chemistry 22, 7215–7224 (2016).

    CAS  PubMed  Google Scholar 

  32. 32.

    Yamamoto, H. et al. Posttranslational processing of FGF23 in osteocytes during the osteoblast to osteocyte transition. Bone 84, 120–130 (2016).

    CAS  PubMed  Google Scholar 

  33. 33.

    Bennett, E. P. et al. Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy. J. Biol. Chem. 274, 25362–25370 (1999).

    CAS  PubMed  Google Scholar 

  34. 34.

    Joshi, H. J. et al. Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases. Glycobiology 28, 284–294 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Minisola, S. et al. Tumour-induced osteomalacia. Nat. Rev. Dis. Prim. 3, 17044 (2017).

    PubMed  Google Scholar 

  36. 36.

    Rafaelsen, S., Johansson, S., Raeder, H. & Bjerknes, R. Long-term clinical outcome and phenotypic variability in hyperphosphatemic familial tumoral calcinosis and hyperphosphatemic hyperostosis syndrome caused by a novel GALNT3 mutation; case report and review of the literature. BMC Genet. 15, 98 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Ramnitz, M. S., Gafni, R. I. & Collins, M. T. Hyperphosphatemic familial tumoral calcinosis. in GeneReviews (R) (eds Adams, M. P. et al.) (Univ. Wash., Seattle, 2018).

  38. 38.

    Takashi, Y. et al. Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation. Proc. Natl Acad. Sci. USA 116, 11418–11427 (2019).

    CAS  PubMed  Google Scholar 

  39. 39.

    Hintze, J. et al. Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines. J. Biol. Chem. 293, 19064–19077 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Wandall, H. H. et al. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. Glycobiology 17, 374–387 (2007).

    CAS  PubMed  Google Scholar 

  41. 41.

    Steentoft, C. et al. A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation. Glycobiology 29, 645–656 (2019).

    PubMed  Google Scholar 

  42. 42.

    Yang, Z. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33, 842–844 (2015).

    CAS  PubMed  Google Scholar 

  43. 43.

    Lonowski, L. A. et al. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. Nat. Protoc. 12, 581–603 (2017).

    CAS  PubMed  Google Scholar 

  44. 44.

    Wandall, H. H. et al. Substrate specificities of three members of the human UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3. J. Biol. Chem. 272, 23503–23514 (1997).

    CAS  PubMed  Google Scholar 

  45. 45.

    Kabsch, W. Xds. Acta Crystallogr. D. 66, 125–132 (2010).

    CAS  PubMed  Google Scholar 

  46. 46.

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

    CAS  PubMed  Google Scholar 

  47. 47.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D. 50, 760–763 (1994).

  48. 48.

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

    PubMed  Google Scholar 

  49. 49.

    Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. 67, 355–367 (2011).

    CAS  PubMed  Google Scholar 

  50. 50.

    Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Maier, J. A. et al. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Plattner, C., Hofener, M. & Sewald, N. One-pot azidochlorination of glycals. Org. Lett. 13, 545–547 (2011).

    CAS  PubMed  Google Scholar 

  54. 54.

    Horcas, I. et al. WSXM: a software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 78, 013705 (2007).

    CAS  PubMed  Google Scholar 

  55. 55.

    Lostao, A., Peleato, M. L., Gomez-Moreno, C. & Fillat, M. F. Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: direct visualization by in situ atomic force microscopy under different redox conditions. Biochim. Biophys. Acta 1804, 1723–1729 (2010).

    CAS  PubMed  Google Scholar 

  56. 56.

    Sun, T., Lin, F. H., Campbell, R. L., Allingham, J. S. & Davies, P. L. An antifreeze protein folds with an interior network of more than 400 semi-clathrate waters. Science 343, 795–798 (2014).

    CAS  PubMed  Google Scholar 

  57. 57.

    Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212–1225 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Tria, G., Mertens, H. D., Kachala, M. & Svergun, D. I. Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCr. J. 2, 207–217 (2015).

    CAS  Google Scholar 

  59. 59.

    Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Bernado, P., Mylonas, E., Petoukhov, M. V., Blackledge, M. & Svergun, D. I. Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem. Soc. 129, 5656–5664 (2007).

    CAS  PubMed  Google Scholar 

Download references


We thank the Diamond Light Source (Oxford) synchrotron beamline I24 (experiment nos. MX14739-6 and MX14739-11) and the SOLEIL synchrotron (Gif-sur-Yvette) SWING beamline (experiment nos. 99170088). We thank ARAID, MEC (grant no. CTQ2013-44367-C2-2-P, BFU2016-75633-P and RTI2018-099592-B-C21), the National Institutes of Health (grant no. GM113534 and instrument grant no. GM113534-01S), the Danish National Research Foundation (grant no. DNRF107), the FCT-Portugal (grant no. UID/Multi/04378/2013) and Gobierno de Aragón (grant nos. E34_R17, E35_17R and LMP58_18) with FEDER (grant no. 2014-2020) funds for ‘Building Europe from Aragón’ for financial support. I.C. thanks the Universidad de La Rioja for the FPI grant. F.M. and H.C. thank FCT-Portugal for IF Investigator (IF/00780/2015), PTDC/BIA-MIB/31028/2017 and UID/Multi/04378/2019 projects, and PTNMR (grant no. ROTEIRO/0031/2013 and PINFRA/22161/2016). P.B. acknowledges support from the Labex EpiGenMed, an ‘Investissements d’avenir’ program (grant no. ANR-10-LABX-12-01). The CBS (Montpellier) is a member of France-BioImaging (FBI, ANR-10-INBS-04-01) and the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INBS-05). The research leading to these results has also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement nos. 283570 and BIOSTRUCTX_5186). We also thank I. Echániz for technical support and K. Moremen from the University of Georgia, Complex Carbohydrate Research Center, for supplying the pGEn2-HsGalNAc-T6 and pGEn2-HsGalNAc-T12 plasmids.

Author information




R.H.-G. designed the crystallization construct and solved the crystal structures. M.R. and R.H.-G. purified the enzymes, crystallized the complexes and refined the crystal structures. I.C. and F.C. synthesized the glycopeptides. F.C. performed the MD simulations. H.Coelho and F.M. performed and analyzed the NMR experiments. T.A.G. and E.J.P.D. performed the kinetic studies together with the Edman amino acid sequencing. Y.N., K.K. and R.M. performed the experiments in cells and did the MALDI–TOF MS mass spectrometry experiments. P.H. and A.L. performed the AFM studies. A.T. and P.B. performed the SAXS experiments. L.C.-L. conducted the expression and purification of GalNAc-T6 and T12 in HEK293 cells. L.H. identified the GalNAc-T3 mutations associated to disease. R.H.-G., T.A.G. and H.Clausen wrote the article with the other authors’ contributions. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ramon Hurtado-Guerrero.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figs 1–17 and Tables 1–6.

Reporting Summary

Supplementary Video 1

A 500 ns MD simulation of TgGalNAc-T3 complexed to UDP-Mn+2 and FGF23c in explicit water

Supplementary Video 2

A 500 ns MD simulation of HsGalNAc-T4 complexed to UDP-Mn+2 and FGF23c in explicit water

Supplementary Video 3

A 500 ns MD simulation of HsGalNAc-T6 complexed to UDP-Mn+2 and FGF23c in explicit water

Supplementary Video 4

A 500 ns MD simulation of HsGalNAc-T12 complexed to UDP-Mn+2 and FGF23c in explicit water

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de las Rivas, M., Paul Daniel, E.J., Narimatsu, Y. et al. Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3. Nat Chem Biol 16, 351–360 (2020).

Download citation

Further reading


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