N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.
N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.
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DOI：
10.1016/j.jbc.2022.101666
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Kizuka Y
中科院分区：
文献类型：
--
作者：
Osuka RF;Hirata T;Nagae M;Nakano M;Shibata H;Okamoto R;Kizuka Y
N-乙酰葡糖胺基转移酶-V（GnT-V或MGAT 5）催化高尔基体中选择性靶蛋白上的N-聚糖β 1，6-GlcNAc分支的形成，并且参与癌症恶性肿瘤和自身免疫性疾病病因学。GnT-V的几个三维结构最近被解决，并阐明了寡糖底物的识别机制。然而，目前尚不清楚GnT-V如何选择性地作用于糖蛋白底物。在这项研究中，我们专注于一个未知的结构域在N-末端侧的管腔区域（N域）的GnT-V，这是以前确定的晶体结构，并旨在揭示其作用的GnT-V行动。使用凝集素印迹和荧光辅助细胞分选分析，我们发现缺乏N结构域的GnT-VΔN突变体在细胞中的生物合成活性受损，这表明N结构域是有效糖基化所必需的。为了阐明这一机制，我们使用HPLC和UDP-Glo测定法测量了纯化的GnT-VΔN对各种底物（寡糖、糖六肽和糖蛋白）的体外活性。令人惊讶的是，GnT-VΔN对糖蛋白底物的活性显著降低，而对寡糖和糖肽底物的活性几乎完全保持不变。最后，GnT-V与底物糖蛋白的对接模型表明，N结构域可以直接与底物多肽相互作用。我们的研究结果表明，GnT-V的N结构域在糖蛋白底物的识别中起着关键作用，为N-聚糖的底物选择性生物合成机制提供了新的见解。
N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan β1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.
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影响因子：
7.5
作者：
Boscher, Cecile;Dennis, James W.;Nabi, Ivan R.
通讯作者：
Nabi, Ivan R.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
11.1
作者：
Kizuka Y;Kitazume S;Fujinawa R;Saito T;Iwata N;Saido TC;Nakano M;Yamaguchi Y;Hashimoto Y;Staufenbiel M;Hatsuta H;Murayama S;Manya H;Endo T;Taniguchi N
通讯作者：
Taniguchi N
影响因子：
12.9
作者：
Darby, John F.;Gilio, Amelia K.;Wu, Liang
通讯作者：
Wu, Liang
影响因子：
2.7
作者：
GU, JG;NISHIKAWA, A;TANIGUCHI, N
通讯作者：
TANIGUCHI, N