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

Boeggeman, Elizabeth and Ramakrishnan, Boopathy and Kilgore, Charlton and Khidekel, Nelly and Hsieh-Wilson, Linda C. and Simpson, John T. and Qasba, Pradman K. (2007) Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method. Bioconjugate Chemistry, 18 (3). pp. 806-814. ISSN 1043-1802. PMCID PMC3534963. doi:10.1021/bc060341n. https://resolver.caltech.edu/CaltechAUTHORS:20150519-130357505

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Abstract

The mutant β1,4-galactosyltransferase (β4Gal-T1), β4Gal-T1-Y289L, in contrast to wild-type β4Gal-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, β4Gal-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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/bc060341nDOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534963PubMed CentralArticle
ORCID:
AuthorORCID
Hsieh-Wilson, Linda C.0000-0001-5661-1714
Additional Information:© 2007 American Chemical Society. Received November 1, 2006; Revised Manuscript Received January 8, 2007; Publication Date (Web): March 20, 2007. We thank Drs. Dimiter S. Dimitrov, Mei-Yun Zhang, and Yang Feng from the Nanobiology program, CCR, NCI, for providing IgG1samples, and Dr. Maria Manzoni from the Structural Glycobiology Section, CCR, NCI, for the critical reading of the manuscript. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government. This Research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Funders:
Funding AgencyGrant Number
National Cancer InstituteUNSPECIFIED
NIHN01-CO-12400
Issue or Number:3
PubMed Central ID:PMC3534963
DOI:10.1021/bc060341n
Record Number:CaltechAUTHORS:20150519-130357505
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150519-130357505
Official Citation:Direct Identification of Nonreducing GlcNAc Residues on N-Glycans of Glycoproteins Using a Novel Chemoenzymatic Method Elizabeth Boeggeman, Boopathy Ramakrishnan, Charlton Kilgore, Nelly Khideke, Linda C. Hsieh-Wilson, John T. Simpson, and Pradman K. Qasba Bioconjugate Chemistry 2007 18 (3), 806-814 DOI: 10.1021/bc060341n
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57661
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:19 May 2015 20:58
Last Modified:10 Nov 2021 21:53

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