Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling

Autor: Coelho, Helena, de las Rivas, Matilde, Grosso, Ana S., Diniz, Ana, Soares, Catia O., Francisco, Rodrigo A., Dias, Jorge S., Companon, Ismael, Sun, Lingbo, Narimatsu, Yoshiki, Vakhrushev, Sergey Y., Clausen, Henrik, Cabrita, Eurico J., Jimenez-Barbero, Jesus, Corzana, Francisco, Hurtado-Guerrero, Ramon, Marcelo, Filipa, 0000-0003-1992-8557, 0000-0001-5324-4998, 0000-0002-9102-3018, 0000-0001-9725-1882, 0000-0003-1428-5695, 0000-0002-3122-9401, 0000-0001-5049-8511
Přispěvatelé: DQ - Departamento de Química, UCIBIO - Applied Molecular Biosciences Unit, European Commission
Rok vydání: 2022
Předmět:
Zdroj: Addi. Archivo Digital para la Docencia y la Investigación
Universidad de Cantabria (UC)
instname
Coelho, H, Rivas, M D L, Grosso, A S, Diniz, A, Soares, C O, Francisco, R A, Dias, J S, Compañon, I, Sun, L, Narimatsu, Y, Vakhrushev, S Y, Clausen, H, Cabrita, E J, Jiménez-Barbero, J, Corzana, F, Hurtado-Guerrero, R & Marcelo, F 2022, ' Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling ', JACS Au, vol. 2, no. 3, pp. 631-645 . https://doi.org/10.1021/jacsau.1c00529
ISSN: 2691-3704
2016-7563
Popis: [EN] The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse gamma-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates. The authors acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding the projects, IF/00780/2015, PTDC/BIA-MIB/31028/2017, and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020), and Associate Laboratory Institute for Health and Bioeconomy─i4HB project (LA/P/0140/2020). The authors also thank FCT-Portugal for the PhD grant attributed to ASG (SFRH/BD/140394/2018) and A.D. (PD/BD/142847/2018) under the PTNMRPhD Program (PD00065/2013) and for the Norma transitória DL 57/2016 Program Contract to J.S.D. The NMR spectrometers are part of the National NMR Facility supported by FCT-Portugal (ROTEIRO/0031/2013-PINFRA/22161/2016, cofinanced by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). RHG thanks ARAID, the Spanish Ministry of Science, Innovation and Universities (BFU2016-75633-P and PID2019-105451GB-I00), and Gobierno de Aragón (E34_R17 and LMP58_18 to R.H-G.) with FEDER (2014–2020) funds for “Building Europe from Aragón” for the financial support. F.C. thanks Agencia Estatal Investigación (AEI, Spain) (grant RTI2018-099592-B-C21). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 956544. J.J.-B. and H.C. thank EU for funding (Tollerant ITN, GA-642157). F.M. and J.J.-B. acknowledge the COST Action GLYCONanoProbes (CA18132). J.J.-B also thanks the European Research Council for the financial support (ERC-2017-AdG, project number 788143-RECGLYCANMR), AEI (Spain, grant RTI218-094751-B-C21), and CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. H.C. acknowledges the Lundbeck Foundation and the Danish National Research Foundation (DNRF107).
Databáze: OpenAIRE
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