Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V

Background: 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 beta 1,6-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.Methods: 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.Results: 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.Conclusions: We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids.General significance: Our data provide new insights into how GnT-V recognizes glycoproteins.

背景：N -糖基化对蛋白质折叠、运输和功能至关重要。N -聚糖以蛋白质选择性的方式具有不同数量的N -乙酰葡糖胺（GlcNAc）分支，由N -乙酰葡糖胺基转移酶 - V（GnT - V或MGAT5）产生的特定蛋白质上的β1,6 -连接的GlcNAc分支促进癌症恶性程度。然而，关于GnT - V如何作用于特定靶蛋白知之甚少。 方法：基于我们的结构模型，我们假设GnT - V与N -聚糖核心或多肽部分以及N -聚糖的接受位点相互作用。为了探索这种可能性，我们选择了四个参与与聚糖核心或周围氨基酸相互作用的候选残基，创建了这些残基的点突变体，并检测了突变体的体外和体内活性。 结果：我们使用包括寡糖和糖蛋白在内的各种类型底物进行的体外酶活性测定显示，V354N突变体对所有测试底物的活性显著降低，底物偏好发生改变，并且K361A对带有天冬酰胺（Asn）的寡糖活性降低，但对没有GlcNAc还原端和Asn的较短寡糖则没有。这些结果表明V354和K361参与对N -聚糖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细胞中糖蛋白修饰的重要性。 结论：我们确定了几个参与GnT - V对N -聚糖核心和周围氨基酸作用的残基。 普遍意义：我们的数据为GnT - V如何识别糖蛋白提供了新的见解。