Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC, CONICET and Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, X5000HUA, Córdoba, Argentina.
Biol Chem. 2020 Aug 27;401(9):1041-1051. doi: 10.1515/hsz-2019-0448.
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.
糖基化是蛋白质中非常常见的翻译后修饰，最近在相关核蛋白上描述了 O-N-乙酰半乳糖胺 (O-GalNAc) 糖基化的起始。在这里，我们评估了 O-GalNAc 聚糖生物合成途径中第二个糖基的核内掺入，以生成末端核心 1 聚糖 (C1G，Galβ3GalNAcαSer/Thr)。使用共聚焦显微镜、酶测定、亲和层析和质谱分析，我们分析了完整细胞、纯化的核和可溶性核质体，以鉴定细胞核中 C1G 生物合成所必需的因素。负责 C1G 合成的酶 C1GalT1 在内核中被检测到，而 C1 半乳糖基转移酶的催化活性存在于核质体和纯化的核中。此外，在完整细胞的核内检测到 C1G，并且还鉴定了暴露 C1G 的核蛋白。这些证据代表了人类细胞核内蛋白质核心 1 O-GalNAc 糖基化的首次证明。这些发现揭示了核蛋白上的一种新的翻译后修饰，在表观遗传学和化学生物学领域具有重要意义。
