RESUMO
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, Galß3GalNAcαSer/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.
Assuntos
Acetilgalactosamina/metabolismo , Núcleo Celular/metabolismo , Glicosilação , HumanosRESUMO
Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of O-GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc -transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.
Assuntos
Acetilgalactosamina/biossíntese , Acetilgalactosamina/química , Núcleo Celular/metabolismo , Polissacarídeos/química , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Glicosilação , Humanos , Lamina Tipo B/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Soybean (Glycine max) is often being cultivated in soils with moderate to high arsenic (As) concentrations or under irrigation with As contaminated groundwater. The purpose of this study was to determine the effect of As on soybean germination, development and nodulation in soybean-Bradyrhizobium japonicum E109 symbiosis, as a first-step approach to evaluate the impact of As on soybean production. Semi-hydroponic assays were conducted using soybean seedlings inoculated and non-inoculated with B. japonicum E109 and treated with arsenate or arsenite. Soybean germination and development, at early stage of growth, were significantly reduced from 10 µM arsenate or arsenite. This also was seen for soybean seedlings inoculated with B. japonicum mainly with arsenite where, in addition, the number of effective nodules was reduced, despite that the microorganism tolerated the metalloid. This minor nodulation could be due to a reduced motility (swarming and swimming) of the microorganism in presence of As. Arsenic concentration in roots was about 250-times higher than in shoots. Transference coefficient values indicated that As translocation to aerial parts was low and As accumulated mainly in roots, without significant differences between inoculated and non-inoculated plants. The presence of As restricted soybean-B. japonicum symbiosis and hence, the efficiency of most used commercial inoculants for soybean. Thus, water and/or soils containing As would negatively impact on soybean production, even in plants inoculated with B. japonicum E109.