Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method

被引:52
作者
Boeggeman, Elizabeth
Ramakrishnan, Boopathy
Kilgore, Charlton
Khidekel, Nelly
Hsieh-Wilson, Linda C.
Simpson, John T.
Qasba, Pradman K.
机构
[1] NCI, Struct Glycobiol Sect, CCR, Nanobiol Program,Ctr Canc Res, Frederick, MD 21702 USA
[2] NCI, Prot Chem Lab, SAIC Inc, Frederick, MD 21702 USA
[3] CALTECH, Div Chem & Chem Engn, Pasadena, CA 91125 USA
[4] CALTECH, Howard Hughes Med Inst, Pasadena, CA 91125 USA
关键词
D O I
10.1021/bc060341n
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:806 / 814
页数:9
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