Abstract
The importance of glycans in various cellular events, especially intracellular and intercellular trafficking of proteins, has been reported in numerous studies. Here, we present a novel method to monitor endocytosis of proteins of interest bearing a specific glycan modification. Using a fluorescence resonance energy transfer technique, we investigated the role of glycan structure on the internalization of insulin-responsive glucose transporter GLUT4. We found that sialylated glycoforms of GFP-tagged GLUT4 appear to be internalized more slowly than non-sialylated GLUT4 upon insulin removal. This novel glycan imaging tool allows probing functional roles of specific glycan modifications in endocytosis of various proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Dube DH, Bertozzi CR (2003) Metabolic oligosaccharide engineering as a tool for glycobiology. Curr Opin Chem Biol 7:616–625
Du J, Meledeo MA, Wang Z et al (2009) Metabolic glycoengineering: sialic acid and beyond. Glycobiology 19:1382–1401
Zaro BW, Yang YY, Hang HC, Pratt MR (2011) Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci U S A 108:8146–8151
Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction. Science 287:2007–2010
Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR (2003) A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc Natl Acad Sci U S A 100:9116–9121
Hsu TL, Hanson SR, Kishikawa K et al (2007) Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci U S A 104:2614–2619
Baskin JM, Prescher JA, Laughlin ST et al (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A 104:16793–16797
Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR (2008) In vivo imaging of membrane-associated glycans in develo** zebrafish. Science 320:664–667
Baskin JM, Dehnert KW, Laughlin ST, Amacher SL, Bertozzi CR (2010) Visualizing envelo** layer glycans during zebrafish early embryogenesis. Proc Natl Acad Sci U S A 107:10360–10365
Dehnert KW, Beahm BJ, Huynh TT et al (2011) Metabolic labeling of fucosylated glycans in develo** zebrafish. ACS Chem Biol 6:547–552
Taniguchi N, Miyoshi E, Gu J, Honke K, Matsumoto A (2006) Decoding sugar functions by identifying target glycoproteins. Curr Opin Struct Biol 16:561–566
Muhlenhoff M, Manegold A, Windfuhr M, Gotza B, Gerardy-Schahn R (2001) The impact of N-glycosylation on the functions of polysialyltransferases. J Biol Chem 276:34066–34073
Wang X, Inoue S, Gu J et al (2005) Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice. Proc Natl Acad Sci U S A 102:15791–15796
Wang X, Gu J, Ihara H et al (2006) Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling. J Biol Chem 281:2572–2577
Partridge EA, Le Roy C, Di Guglielmo GM et al (2004) Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis. Science 306:120–124
Ohtsubo K, Takamatsu S, Minowa MT et al (2005) Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes. Cell 123:1307–1321
Kitazume S, Imamaki R, Ogawa K et al (2010) Alpha2,6-sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signaling. J Biol Chem 285:6515–6521
Lau KS, Partridge EA, Grigorian A et al (2007) Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 129:123–134
Haga Y, Ishii K, Hibino K et al (2012) Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer. Nat Commun 3:907
Acknowledgments
This work was partly supported by a Grant-in-Aid for Exploratory Research (25650041) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. S.) and a Grant-in-Aid for JSPS postdoctoral fellow (to Y. H.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Haga, Y., Suzuki, T. (2014). Use of Transmembrane FRET to Investigate the Internalization of Glycosylated Proteins. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0944-5_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0943-8
Online ISBN: 978-1-4939-0944-5
eBook Packages: Springer Protocols