期刊
JACS AU
卷 2, 期 3, 页码 631-645出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacsau.1c00529
关键词
49-GalNAc glycosylation; GalNAc-Ts; mucin-1; NMR; molecular dynamics; cell analysis
资金
- Fundacao para a Ciencia e a Tecnologia (FCT-Portugal) [IF/00780/2015, PTDC/BIA-MIB/31028/2017, UIDP/04378/2020, UIDB/04378/2020, LA/P/0140/2020]
- FCT-Portugal [SFRH/BD/140394/2018, PD/BD/142847/2018, PD00065/2013, DL 57/2016]
- FCT-Portugal (FEDER through COMPETE 2020) [ROTEIRO/0031/2013PINFRA/22161/2016]
- FCT-Portugal (FEDER through POCI) [ROTEIRO/0031/2013PINFRA/22161/2016]
- FCT-Portugal (FEDER through PORL) [ROTEIRO/0031/2013PINFRA/22161/2016]
- FCT-Portugal (FCT through PIDDAC) [ROTEIRO/0031/2013PINFRA/22161/2016]
- ARAID
- Spanish Ministry of Science, Innovation and Universities [BFU2016-75633-P, PID2019-105451GB-I00]
- Gobierno de Aragon [E34_R17, LMP58_18]
- FEDER (20142020) funds for Building Europe from Aragon
- Agencia Estatal Investigacion (AEI, Spain) [RTI2018-099592-B-C21]
- European Union [956544]
- EU [GA-642157]
- COST Action GLYCONanoProbes [CA18132]
- European Research Council (ERC-2017-AdG) [788143-RECGLYCANMR]
- AEI (Spain) [RTI218-094751-B-C21]
- CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain
- Danish National Research Foundation [DNRF107]
- Fundação para a Ciência e a Tecnologia [PTDC/BIA-MIB/31028/2017, SFRH/BD/140394/2018, PD/BD/142847/2018] Funding Source: FCT
This study uses NMR spectroscopy and molecular modeling techniques to reveal the mechanisms of GalNAc-T2/T3/T4 isoforms in MUC1 O-glycosylation. The experiments show that the lectin domain plays an important role in site selection, and neighboring tandem repeats are critical for subsequent glycosylation. Additionally, specific salt bridges and inverse gamma-turn conformations in MUC1 are found to be key structural motifs for GalNAc-T4 specificity. In-cell analysis also demonstrates that GalNAc-T4 is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo.
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 GaINAc 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.
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