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Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V.
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-09-02 , DOI: 10.1016/j.bbagen.2020.129726
Tetsuya Hirata 1 , Masamichi Nagae 2 , Reina F Osuka 3 , Sushil K Mishra 4 , Mayumi Yamada 5 , Yasuhiko Kizuka 1
Affiliation  

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 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.

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.



中文翻译:

N-乙酰氨基葡糖基转移酶-V对聚糖和蛋白质底物的识别。

背景

N-糖基化对于蛋白质折叠,运输和功能至关重要。N-聚糖以蛋白质选择性方式具有不同数量的N-乙酰氨基葡萄糖(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分支促进癌症恶性肿瘤。然而,关于GnT-V如何作用于特定靶蛋白的了解甚少。

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-聚糖的受体位点相互作用。为了探索这种可能性,我们选择了四个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,并研究了该突变体的体外体内活性。

结果

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突变体对所有被测底物的活性均显着降低,而底物偏好有所改变,而K361A对具有天冬酰胺(Asn)的寡糖的活性却有所降低,但没有没有GlcNAc和Asn还原末端的较短的寡糖。这些结果表明,V354和K361参与了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HeLa细胞进行了抢救实验,并证实了这些残基对于修饰细胞中糖蛋白的重要性。

结论

我们确定了一些参与GnT-V对N-聚糖核心和周围氨基酸的作用的残基。

一般意义

我们的数据为GnT-V如何识别糖蛋白提供了新的见解。

更新日期:2020-09-10
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