Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-41345 Gothenburg, Sweden.
Mol Omics. 2020 Jun 1;16(3):243-257. doi: 10.1039/c9mo00175a. Epub 2020 Apr 8.
The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of I-LDN-BSA to various clinical H. pylori isolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a β1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a I-labelled LDN-BSA neoglycoconjugate to clinical H. pylori isolates was identified.
β4-N-乙酰半乳糖胺基转移酶 3（B4GALNT3）将 GalNAc 通过β1,4 键连接到 GlcNAc 上，在寡糖上形成 LacdiNAc（LDN）决定簇。已经提出 LacdiNAc 结合黏附素（LabA）通过与 LDN 决定簇结合介导幽门螺杆菌附着在胃黏膜上。B4GALNT3 对 O-聚糖核心 2、核心 3 和扩展核心 1 前体中 GlcNAc 残基的 O-聚糖核心链特异性定义较差。我们使用 CHO-K1 细胞的瞬时转染和粘蛋白型免疫球蛋白融合蛋白作为报告蛋白，研究了 B4GALNT3 在 O-聚糖核心 2、核心 3 和扩展核心 1 前体中的 GlcNAc 残基上的特异性。在微量滴定板结合测定中评估了 LabA 阳性 H. pylori J99 和 26695 菌株与携带不同 O-聚糖核心链上的 LDN 决定簇的粘蛋白融合蛋白以及带有和不带 LDN 的人胃粘蛋白之间的结合，同时在溶液中评估了 I-LDN-BSA 与各种临床 H. pylori 分离株的结合。液相色谱-串联质谱（LC-MS/MS）和 Western blot 证实了 B4GALNT3 活性需要末端 GlcNAc。B4GALNT3 将β1,4 连接的 GalNAc 添加到 GlcNAc 上，无论后者是由核心 2、核心 3 还是扩展核心 1 链携带。在微量滴定板结合测定中，未检测到 H. pylori 菌株 26 695 和 J99 对胃粘蛋白或携带核心 2、3 和扩展核心 1 O-聚糖的粘蛋白型融合蛋白上的 LDN 决定簇的 LDN 介导的粘附，也未鉴定出 I 标记的 LDN-BSA 新糖缀合物与临床 H. pylori 分离株的结合。
