Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling.
Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling.
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DOI：
10.1021/acs.bioconjchem.0c00454
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发表时间：
2020-09-16
影响因子：
4.7
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Wu ZL;Luo A;Grill A;Lao T;Zou Y;Chen Y
O-GlcNAc酰化是细胞溶质和核蛋白上的可逆丝氨酸/苏氨酸糖基化，其参与各种调节途径。然而，O-GlcNAc化底物的检测和定量一直具有挑战性。在这里，我们报告了一种高效的方法，用于通过串联聚糖标记，其中O-GlcNAc的半乳糖基化，然后唾液酸化与荧光团共轭的唾液酸残基，从而实现高灵敏度的荧光检测的O-GlcNAc修饰的识别。该方法在已知通过O-GlcNAc化修饰的各种蛋白质（包括CK 2、NOD 2、SREBP 1c、AKT 1、PKM和PFKFB 3）以及HEK 293细胞的核提取物上进行了验证。使用这种方法，我们然后报告的证据表明，缺氧诱导因子HIF 1 α是一个潜在的目标O-GlcNAc酰化，这表明代谢O-GlcNAc途径和缺氧途径之间可能有直接的联系。
O-GlcNAcylation is a reversible serine/threonine glycosylation on cytosolic and nuclear proteins that are involved in various regulatory pathways. However, the detection and quantification of O-GlcNAcylation substrates have been challenging. Here, we report a highly efficient method for the identification of O-GlcNAc modification via tandem glycan labeling, in which O-GlcNAc is first galactosylated and then sialylated with a fluorophore-conjugated sialic acid residue, therefore enabling highly sensitive fluorescent detection. The method is validated on various proteins that are known to be modified by O-GlcNAcylation including CK2, NOD2, SREBP1c, AKT1, PKM, and PFKFB3, and on the nuclear extract of HEK293 cells. Using this method, we then report the evidence that hypoxia-inducible factor HIF1α is a potential target for O-GlcNAcylation, suggesting a possibly direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.
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影响因子：
8
作者：
Sodi VL;Bacigalupa ZA;Ferrer CM;Lee JV;Gocal WA;Mukhopadhyay D;Wellen KE;Ivan M;Reginato MJ
通讯作者：
Reginato MJ
影响因子：
56.9
作者：
Jaakkola, P;Mole, DR;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
64.8
作者：
Maxwell, PH;Wiesener, MS;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
16
作者：
Ferrer, Christina M.;Lynch, Thomas P.;Sodi, Valerie L.;Falcone, John N.;Schwab, Luciana P.;Peacock, Danielle L.;Vocadlo, David J.;Seagroves, Tiffany N.;Reginato, Mauricio J.
通讯作者：
Reginato, Mauricio J.
DOI：
10.1073/pnas.072072399
发表时间：
2002-04-16
影响因子：
11.1
作者：
Vosseller, K;Wells, L;Hart, GW
通讯作者：
Hart, GW