Abstract
Glycosylation is a very frequent post-translational modification in proteins, and the initiation of O-N-acetylgalactosamine (O-GalNAc) glycosylation has been recently described on relevant nuclear proteins. Here we evaluated the nuclear incorporation of a second sugar residue in the biosynthesis pathway of O-GalNAc glycans to yield the terminal core 1 glycan (C1G, Galβ3GalNAcαSer/Thr). Using confocal microscopy, enzymatic assay, affinity chromatography, and mass spectrometry, we analyzed intact cells, purified nuclei and soluble nucleoplasms to identify the essential factors for C1G biosynthesis in the cell nucleus. The enzyme C1GalT1 responsible for C1G synthesis was detected inside the nucleus, while catalytic activity of C1Gal-transferase was present in nucleoplasm and purified nuclei. In addition, C1G were detected in the nucleus inside of intact cells, and nuclear proteins exposing C1G were also identified. These evidences represent the first demonstration of core 1 O-GalNAc glycosylation of proteins in the human cell nucleus. These findings reveal a novel post-translational modification on nuclear proteins, with relevant repercussion in epigenetic and chemical biology areas.
Funding source: CONICET
Award Identifier / Grant number: PIP 11220150100226
Funding source: ANPCyT
Funding source: FONCyT
Award Identifier / Grant number: PICT 2018-03228
Funding source: MinCyT
Funding source: SeCyT, UNC
Funding source: Mizutani Foundation for Glycoscience
Award Identifier / Grant number: 200062
Acknowledgments
This study was supported by funding (to F.J.I.) from CONICET (PIP 11220150100226); ANPCyT, FONCyT (PICT 2018-03228), MinCyT, Pcia Cba (PID 48 Res. 144/18); and SeCyT, UNC in Argentina; and Mizutani Foundation for Glycoscience (No. 200062), Japan. The authors are grateful to S. Deza and G. Schachner for cell culture assistance, and Drs. C. Mas and C. Sampedro for confocal microscopy assistance. Y.C.G. has fellowship assistance from CONICET. R.D.L. and F.J.I. are Career Investigators of CONICET.
Conflict of interest statement: The authors declare no conflict of interest.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2019-0448).
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