Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method
Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method
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DOI：
10.1021/bc060341n
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发表时间：
2007-05-01
影响因子：
4.7
通讯作者：
Qasba, Pradman K.
中科院分区：
文献类型：
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作者：
Boeggeman, Elizabeth;Ramakrishnan, Boopathy;Qasba, Pradman K.
与野生型β 4Gal-T1相比，突变型β 4Gal-T1-Y289 L能够将GalNAc从糖供体UDP-GalNAc转移到受体GlcNAc，其效率与半乳糖从UDP-Gal转移的效率一样好。此外，该突变体还可以将修饰的糖C2酮基半乳糖从其UDP衍生物转移到蛋白质上的O-GlcNAc修饰，这为开发用于检测蛋白质上的O-GlcNAc翻译后修饰的高度灵敏的化学酶促方法提供了功能手柄。我们在此报告，修饰的糖，C2酮基半乳糖，可以转移到N-连接的糖蛋白，如卵清蛋白或无唾液酸-agalacto-IgG 1上的游离GlcNAc残基。该转移严格依赖于突变酶和半乳糖的酮衍生物两者的存在。此外，裂解N-连接寡糖链的糖蛋白的PNGase F处理表明，修饰的糖已转移至糖蛋白的N-聚糖链，而不是蛋白质部分。应用突变半乳糖基转移酶，β 4Gal-T1-Y289 L，产生糖缀合物携带糖部分与反应基团，证明。我们设想了这种技术的广泛潜力，例如通过聚糖链将货物分子连接到糖蛋白（例如单克隆抗体）的可能性，从而有助于将药物糖靶向到作用位点或用作生物探针。
The mutant beta 1,4-galactosyltransferase (beta 4Gal-T1), beta 4Gal-T1-Y289L, in contrast to wild-type beta 4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta 4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.