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.
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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.
The authors declare no competing interests.
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Supplementary Figs 1–17 and Tables 1–6.
A 500 ns MD simulation of TgGalNAc-T3 complexed to UDP-Mn+2 and FGF23c in explicit water
A 500 ns MD simulation of HsGalNAc-T4 complexed to UDP-Mn+2 and FGF23c in explicit water
A 500 ns MD simulation of HsGalNAc-T6 complexed to UDP-Mn+2 and FGF23c in explicit water
A 500 ns MD simulation of HsGalNAc-T12 complexed to UDP-Mn+2 and FGF23c in explicit water
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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 (2020). https://doi.org/10.1038/s41589-019-0444-x