Glycan Array Synthesis Core D, Consortium for Functional Glycomics, The Scripps Research Institute, La Jolla, CA 92037, USA.
Glycobiology. 2012 Nov;22(11):1453-64. doi: 10.1093/glycob/cws101. Epub 2012 Jul 11.
Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans.
多糖 N-乙酰乳糖胺延伸物在 N-和 O-连接糖链上的结构越来越被认为是具有重要生物学意义的，但获取这些结构的方法并不广泛。在这里，我们报告了来自幽门螺杆菌的细菌β3-N-乙酰氨基葡萄糖基转移酶（β3GlcNAcT）的详细底物特异性和催化效率，该酶可适应具有多聚-LacNAc 延伸的丰富多样聚糖的合成。这种糖基转移酶对线性和支化聚糖上的 1 型、2 型、3 型和 4 型半乳糖基基序的接受体具有惊人的广泛特异性，这些基序常见于 N-连接、O-连接和 I-抗原糖链上。这一发现使具有聚糖结合研究用复杂配体的生产成为可能。尽管该酶对 2 型（Galβ1-4GlcNAc）接受体表现出优先活性，但它能够将 N-乙酰氨基葡萄糖（GlcNAc）以β1-3 键连接到 1 型（Galβ1-3GlcNAc）或 3/4 型（Galβ1-3GalNAcα/β）序列上。因此，通过交替使用生成β1-4 或β1-3 键的半乳糖基转移酶与 H. pyloriβ3GlcNAcT，可以用 2 型和 1 型 LacNAc 重复序列延伸各种 N-连接、O-连接和 I-抗原接受体。最后，在尿苷二磷酸（UDP）-GlcNAc 和 UDP-Gal 的存在下，将二-LacNAc 双触角 N-糖肽与β3GlcNAcT 和 GalT-1 一锅温育，在产物中原先的基础上增加了 15 个额外的 LacNAc 单元，这在基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析中表现为代表 GlcNAc 和 Gal 残基的替代添加的一系列顺序离子峰。总体而言，我们的数据表明 H. pyloriβ3GlcNAcT 的底物特异性比以前认识到的更广泛，并证明其作为复杂聚糖的制备化学酶合成的有效资源的能力。
