Molecular basis for fibroblast growth factor 23 o-glycosylation by galnac-t3

ABSTRACT 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 through your institution Buy or subscribe This is a preview of subscription content, access via

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Article Open access 01 May 2023 MOLECULAR STRUCTURE AND ENZYMATIC MECHANISM OF THE HUMAN COLLAGEN HYDROXYLYSINE GALACTOSYLTRANSFERASE GLT25D1/COLGALT1 Article Open access 16 April 2025

GLYCOSYLTRANSFERASE 8 DOMAIN-CONTAINING PROTEIN 1 (GLT8D1) IS A UDP-DEPENDENT GALACTOSYLTRANSFERASE Article Open access 07 December 2023 DATA AVAILABILITY The crystal structures of

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flexible proteins using small-angle X-ray scattering. _J. Am. Chem. Soc._ 129, 5656–5664 (2007). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS 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 Author notes * These authors contributed equally: Matilde de las Rivas, Earnest James Paul Daniel, Yoshiki

Narimatsu, Ismael Compañón. AUTHORS AND AFFILIATIONS * BIFI, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza, Spain Matilde de las Rivas, Laura

Ceballos-Laita & Ramon Hurtado-Guerrero * Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Earnest James Paul Daniel & Thomas A. Gerken * Copenhagen

Center for Glycomics, Department of Cellular and Molecular Medicine, School of Dentistry, University of Copenhagen, Copenhagen, Denmark Yoshiki Narimatsu, Kentaro Kato, Lars Hansen, Henrik

Clausen & Ramon Hurtado-Guerrero * Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, Logroño, Spain Ismael Compañón & Francisco Corzana *

Department of Eco-epidemiology, Institute of Tropical Medicine Nagasaki University, Nagasaki, Japan Kentaro Kato * Laboratorio de Microscopías Avanzadas, Instituto de Nanociencia de Aragón,

Universidad de Zaragoza, Zaragoza, Spain Pablo Hermosilla & Anabel Lostao * Swing Beamline, Synchrotron SOLEIL, Gif sur Yvette, France Aurélien Thureau * UCIBIO, REQUIMTE, Departamento

de Química, Faculdade de Ciências e Tecnologia, Universidade de Nova de Lisboa, Caparica, Portugal Helena Coelho & Filipa Marcelo * CIC bioGUNE, Bizkaia Technology Park, Derio, Spain

Helena Coelho * Centre de Biochimie Structurale. INSERM, CNRS, Université de Montpellier, Montpellier, France Pau Bernadó * Department of Hematology, Graduate School of Medicine, Kyoto

University, Kyoto, Japan Ryota Maeda * Fundación ARAID, Zaragoza, Spain Anabel Lostao & Ramon Hurtado-Guerrero * Instituto de Ciencia de Materiales de Aragón, Universidad de

Zaragoza-CSIC, Zaragoza, Spain Anabel Lostao Authors * Matilde de las Rivas View author publications You can also search for this author inPubMed Google Scholar * Earnest James Paul Daniel

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can also search for this author inPubMed Google Scholar * Pau Bernadó View author publications You can also search for this author inPubMed Google Scholar * Filipa Marcelo View author

publications You can also search for this author inPubMed Google Scholar * Lars Hansen View author publications You can also search for this author inPubMed Google Scholar * Ryota Maeda View

author publications You can also search for this author inPubMed Google Scholar * Anabel Lostao View author publications You can also search for this author inPubMed Google Scholar *

Francisco Corzana View author publications You can also search for this author inPubMed Google Scholar * Henrik Clausen View author publications You can also search for this author inPubMed 

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for this author inPubMed Google Scholar CONTRIBUTIONS 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 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.

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