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.

Download full-text


Available from: Christopher Taron, Apr 21, 2015
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: GPI-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid glycosylphosphatidylinositol (GPI). GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroys cell integrity. Here we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the non-destructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.This article is protected by copyright. All rights reserved
    Proteomics 11/2014; 14(21-22). DOI:10.1002/pmic.201400148 · 3.81 Impact Factor
  • Chapter: References

    Bioconjugate Techniques (Third edition), Third edit edited by Hermanson, Greg T, 01/2013: pages 1017-1094; Academic Press., ISBN: 9780123822390
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bioconjugate Techniques, 3rd Edition, is the essential guide to the modification and cross linking of biomolecules for use in research, diagnostics, and therapeutics. It provides highly detailed information on the chemistry, reagent systems, and practical applications for creating labeled or conjugate molecules. It also describes dozens of reactions, with details on hundreds of commercially available reagents and the use of these reagents for modifying or crosslinking peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and synthetic polymers. Offers a one-stop source for proven methods and protocols for synthesizing bioconjugates in the lab. Provides step-by-step presentation makes the book an ideal source for researchers who are less familiar with the synthesis of bioconjugates. Features full color illustrations. Includes a more extensive introduction into the vast field of bioconjugation and one of the most thorough overviews of immobilization chemistry ever presented.
Show more