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Fluorogenic probes reveal a role of GLUT4 N-glycosylation in intracellular trafficking

Nature Chemical Biology volume 12, pages 853859 (2016) | Download Citation

Abstract

Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.

  • Compound

    (E)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((2-(2-(2-((2-(3-((4-(2-((3,5-dinitrobenzyl)amino)-2-oxoethyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxybenzyl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)benzoate

  • Compound

    (E)-(11-(1-(2-(3-((4-(2-((3,5-dinitrobenzyl)amino)-2-oxoethyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxyphenyl)-12-oxo-2,5,8-trioxa-11-azapentadecan-15-yl)-1-ethyl-10,10-dimethyl-3,4,8,9,10,11-hexahydro-2H-dipyrido[3,2-b:2′,3′-i]phenoxazin-1-ium-8-yl)methanesulfonate

  • Compound

    (E)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((2-(2-(2-((2-(3-((4-((3,5-dinitrobenzyl)carbamoyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxybenzyl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)benzoate

  • Compound

    (E)-(11-(1-(2-(3-((4-((3,5-dinitrobenzyl)carbamoyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxyphenyl)-12-oxo-2,5,8-trioxa-11-azapentadecan-15-yl)-1-ethyl-10,10-dimethyl-3,4,8,9,10,11-hexahydro-2H-dipyrido[3,2-b:2′,3′-i]phenoxazin-1-ium-8-yl)methanesulfonate

  • Compound

    (E)-4-((2-(2-(2-((2-(3-((4-((3,5-dinitrobenzyl)carbamoyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxybenzyl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

  • Compound

    (E)-6-((2-(2-(2-((2-(3-((4-((3,5-dinitrobenzyl)carbamoyl)phenyl)thio)-3-oxoprop-1-en-1-yl)-5-hydroxybenzyl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′-diyl diacetate

  • Compound

    N-(3,5-dinitrobenzyl)acetamide

  • Compound

    N-(4-nitrobenzyl)acetamide

  • Compound

    2-(4-(tritylthio)phenyl)acetic acid

  • Compound

    N-(3,5-dinitrobenzyl)-2-(4-(tritylthio)phenyl)acetamide

  • Compound

    N-(3,5-dinitrobenzyl)-2-(4-mercaptophenyl)acetamide

  • Compound

    4-(tritylthio)benzoic acid

  • Compound

    N-(3,5-dinitrobenzyl)-4-(tritylthio)benzamide

  • Compound

    N-(3,5-dinitrobenzyl)-4-mercaptobenzamide

  • Compound

    methyl (E)-3-(2-(14,14-dimethyl-12-oxo-2,5,8,13-tetraoxa-11-azapentadecyl)-4-(methoxymethoxy)phenyl)acrylate

  • Compound

    S-(4-(2-((3,5-dinitrobenzyl)amino)-2-oxoethyl)phenyl)(E)-3-(2-(14,14-dimethyl-12-oxo-2,5,8,13-tetraoxa-11-azapentadecyl)-4-(methoxymethoxy)phenyl)prop-2-enethioate

  • Compound

    S-(4-((3,5-dinitrobenzyl)carbamoyl)phenyl)(E)-3-(2-(14,14-dimethyl-12-oxo-2,5,8,13-tetraoxa-11-azapentadecyl)-4-(methoxymethoxy)phenyl)prop-2-enethioate

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Acknowledgements

This research was supported by JST, PRESTO, MEXT of Japan (grants 25220207, 26102529, 15K12754 to K.K.; 26282215 to Y.H.; and 14J00755 to S.H.), CREST of JST (K.K.), the Asahi Glass Foundation (K.K.), the Uehara Memorial Foundation (K.K.), the Naito Foundation (Y.H.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Y.H.), and the Program for Creating Future Wisdom, Osaka University, selected in 2014 (Y.H.). We thank Y. Haga (Japanese Foundation for Cancer Research, Tokyo, Japan) for valuable suggestions and the gift of GLUT4 plasmids, and M. Nishiura for experimental support.

Author information

Affiliations

  1. Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.

    • Shinya Hirayama
    • , Yuichiro Hori
    • , Zsolt Benedek
    •  & Kazuya Kikuchi
  2. Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.

    • Yuichiro Hori
    •  & Kazuya Kikuchi
  3. JST, PRESTO, Suita, Osaka, Japan.

    • Yuichiro Hori
  4. Glycometabolome Team, RIKEN Global Research Cluster, Wako, Saitama, Japan.

    • Tadashi Suzuki

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Contributions

S.H., Y.H., T.S. and K.K. designed experiments. S.H. and Z.B. synthesized and characterized chemical probes and performed the cell experiment. T.S. cloned and provided GLUT4 gene. S.H., Y.H., T.S. and K.K. wrote the manuscript. Y.H. and K.K. designed the project. All of the authors contributed to the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kazuya Kikuchi.

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    Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–28.

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DOI

https://doi.org/10.1038/nchembio.2156