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Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase

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Abstract

Glycosphingolipids, particularly gangliosides, are known to modulate growth factor receptor tyrosine kinase. A well-documented example is the inhibitory effect of GM3 on kinase associated with epidermal growth factor receptor (EGFR) in human epidermoid carcinoma A431 cells. Lyso-GM3 was detected as a minor component in A431 cells, and may function as an auxiliary factor in GM3-dependent inhibition of EGFR. We studied the inhibitory effect of chemically synthesized GM3, lyso-GM3, and its derivatives, on EGFR function, based on their interaction in membrane microdomain, with the following major findings: (1) GM3, EGFR, and caveolin coexist, but tetraspanins CD9 and CD82 are essentially absent, within the same low-density membrane fraction, separated by sucrose density gradient ultracentrifugation. (2) Strong interaction between EGFR and GM3 was indicated by increasing binding of EGFR to GM3-coated polystyrene beads, in a GM3 dose-dependent manner. Confocal microscopy results suggested that three components in the microdomain (GM3, EGFR, and caveolin) are closely associated. (3) Lyso-GM3 or lyso-GM3 dimer strongly inhibited EGFR kinase activity, in a dose-dependent manner, while lyso-GM3 trimer and tetramer did not. >50 μM lyso-GM3 was cytolytic, while >50 μM lyso-GM3 dimer was not cytolytic, yet inhibited EGFR kinase strongly. Thus, lyso-GM3 and its dimer exert an auxiliary effect on GM3-induced inhibition of EGFR kinase and cell growth, and lyso-GM3 dimer may be a good candidate for pharmacological inhibitor of epidermal tumor growth.

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Abbreviations

DMEM:

Dulbecco’s modified Eagle’s medium

EGF:

epidermal growth factor

EGFR:

epidermal growth factor receptor

FBS:

fetal bovine serum

GFR:

growth factor receptor

GM3:

NeuAcα3Galβ4Glcβ1Cer

HPTLC:

high-performance thin-layer chromatography

HRP:

horseradish peroxidase

mAb:

monoclonal antibody

MALDI-TOF MS:

matrix assisted laser desorption/ionization time-of-flight mass spectrometry

PBS:

phosphate-buffered saline

PNF:

post-nuclear fraction

r.t.:

room temperature

TBS:

Tris-buffered saline

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Acknowledgements

This study was supported in part by NIH/ National Cancer Institute grant R01 CA080054 to SH.

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Authors and Affiliations

  1. Pacific Northwest Research Institute, University of Washington, Seattle, WA, 98122, USA

    Yoshimi Murozuka & Sen-itiroh Hakomori

  2. Department of Pathobiology, University of Washington, Seattle, WA, 98195, USA

    Yoshimi Murozuka & Sen-itiroh Hakomori

  3. Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

    Yoshimi Murozuka & Kenichi Hatanaka

  4. Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatuta-cho, Midori-ku, Yokohama, 226-8501, Japan

    Naoko Watanabe

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  1. Yoshimi Murozuka
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  2. Naoko Watanabe
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  4. Sen-itiroh Hakomori
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Correspondence to Kenichi Hatanaka or Sen-itiroh Hakomori.

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Murozuka, Y., Watanabe, N., Hatanaka, K. et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj J 24, 551–563 (2007). https://doi.org/10.1007/s10719-007-9051-2

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