O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and the prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the tetratricopeptide repeat region recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.
中文翻译：
O-GlcNAc 转移酶 (OGT) 是一种必需的糖基化酶，可催化 N-乙酰葡糖胺添加到核蛋白和细胞质蛋白的丝氨酸或苏氨酸残基上。该酶糖基化范围广泛的肽序列，糖基化位点的预测已被证明具有挑战性。缺乏一组经过实验验证的未被 OGT 糖基化的多肽序列，这使得预测合法糖基化位点变得更加困难。在这里，我们测试了一些本质上无序的蛋白质区域作为 OGT 的底物，以建立一组未被 OGT 糖基化的序列。阴性数据集表明 OGT 目标存在氨基酸组成偏差。通过修改融合在肉瘤 (FUS) 中的蛋白质的氨基酸组成以增强糖基化，验证了这种组成偏差。NMR 实验证明 OGT 的四肽重复区域可以结合 FUS，并且促进糖基化的突变增强了结合。这些结果提供证据表明，四肽重复区域识别具有特定组成的底物的无序片段以促进糖基化，从而提供对 OGT 广泛特异性的洞察。
