In vivo incorporation of an azide-labeled sugar analog to detect mammalian glycosylphosphatidylinositol molecules isolated from the cell surface

New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Carbohydrate research (Impact Factor: 1.93). 09/2012; 362C:62-69. DOI: 10.1016/j.carres.2012.09.012
Source: PubMed


N-Acetylgalactosamine (GalNAc) linked to the first mannose of glycosylphosphatidylinositol (GPI) core has been previously reported to be heterogeneously present on some mammalian GPI-anchored proteins. Here we present a method for profiling GalNAc-containing GPI-anchored proteins in mammalian cells by metabolic labeling with tetraacetylated N-azidoacetylgalactosamine (GalNAz) followed by biotinylation of the incorporated sugar analog. We have labeled both endogenous and recombinant GPI-anchored proteins with GalNAz, and demonstrated that the azide-activated sugar gets incorporated into the GPI glycan, likely as an unsubstituted side branch of the core structure. GalNAz was detected only on GPI molecules attached to proteins, and not on GPI precursors, indicating that GalNAc modification takes place after the GPI anchor is transferred to protein. We have highlighted the utility of this cell labeling approach by demonstrating the ability to examine specific GalNAc-containing GPI-anchored proteins isolated non-destructively from separate membrane domains (apical and basolateral) in polarized epithelial cells. This study represents the first demonstration of site-specific in vivo labeling of a GPI moiety with a synthetic sugar analog.

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Available from: Christopher Taron, Apr 21, 2015
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    • "The first method (sugar analog capture enrichment ) consisted of metabolically incorporating the azido sugar analog GalNAz into cellular glycans prior to the PI- PLC-mediated release of GPI-APs (Fig. 1B). Previous studies had demonstrated that GalNAz (and its epimerized N-azidoacetylglucosamine form) becomes incorporated into N-and O-linked glycans as well as into some GPI anchors [36] [45] [46]. Hence, GPI-APs from labeled cells may contain the sugar analog in multiple glycans (Fig. 1A). "
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