Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu 501-1193, Japan; Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan.
Department of Molecular Immunology, Research Institute for Microbial Diseases, and Laboratory of Molecular Immunology, Immunology Frontier Research Center (iFReC), Osaka University, Suita, Osaka 565-0871, Japan.
Biochim Biophys Acta Gen Subj. 2020 Dec;1864(12):129726. doi: 10.1016/j.bbagen.2020.129726. Epub 2020 Sep 2.
N-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the β1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins.
Based on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants.
Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells.
We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids.
Our data provide new insights into how GnT-V recognizes glycoproteins.
N-糖基化对于蛋白质折叠、运输和功能至关重要。N-糖链以蛋白质选择性的方式具有不同数量的 N-乙酰葡萄糖胺（GlcNAc）分支，并且 N-乙酰氨基葡萄糖基转移酶-V（GnT-V 或 MGAT5）产生的特定蛋白质上的β1,6 连接的 GlcNAc 分支促进了癌症的恶性程度。然而，人们对 GnT-V 如何作用于特定靶蛋白知之甚少。
基于我们的结构模型，我们假设 GnT-V 与 N-糖链核心或多肽部分以及 N-糖链的受体部位相互作用。为了探索这种可能性，我们选择了涉及与聚糖核心或周围氨基酸相互作用的四个候选残基，创建了这些残基的点突变体，并检查了突变体的体外和体内活性。
我们使用各种类型的底物（包括寡糖和糖蛋白）进行的体外酶分析表明，V354N 突变体对所有测试底物的活性均显著降低，且具有改变的底物偏好，而 K361A 对具有天冬酰胺（Asn）的寡糖的活性降低，但对没有 GlcNAc 和 Asn 的还原末端的较短寡糖的活性没有降低。这些结果表明 V354 和 K361 参与了 N-糖链核心和周围氨基酸的识别。我们进一步使用 GnT-V 敲除的 HeLa 细胞进行了挽救实验，并证实了这些残基对于细胞中糖蛋白修饰的重要性。
我们确定了几个涉及 GnT-V 对 N-糖链核心和周围氨基酸作用的残基。
我们的数据为 GnT-V 识别糖蛋白的机制提供了新的见解。
