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Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

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Abstract

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.

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Abbreviations

GalCer:

Galactosylceramide

LacCer:

Lactosylceramide

GalCerS:

GalCer synthase

GM3/GM4S:

GM3/GM4 synthase

ER:

Endoplasmic reticulum

GlcCer:

Glucosylceramide

GlcCerS:

GlcCer synthase

LacCerS:

LacCer synthase

TLC:

Thin-layer chromatography

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Acknowledgments

We thank Dr. A. Sweeney for critical reading of this manuscript, Dr. M. Ito for providing RPMI 1846 cells and Dr. Y. Hirabayashi for providing GM-95 cells. This work was supported by Grant-in-Aid for Scientific Research (B; 23370064) (J.I.) and Mizutani Glycoscience Foundation (J.I.).

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Authors and Affiliations

  1. Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan

    Satoshi Uemura, Shinji Go, Fumi Shishido & Jin-ichi Inokuchi

  2. Molecular Genetic Research Department of Chemistry and Biological Science College of Science, Engineering Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, 252-5258, Japan

    Satoshi Uemura

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  1. Satoshi Uemura
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  2. Shinji Go
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  3. Fumi Shishido
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  4. Jin-ichi Inokuchi
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Correspondence to Satoshi Uemura or Jin-ichi Inokuchi.

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Highlights

- First establishment of GM4-reconstituted cells by overexpression of ST3Gal5 gene.

- No competition between the acceptor substrates LacCer and GalCer for GM3 and GM4 synthesis.

- Expression machinery of GM4 is independent from that of GM3.

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Uemura, S., Go, S., Shishido, F. et al. Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells. Glycoconj J 31, 101–108 (2014). https://doi.org/10.1007/s10719-013-9499-1

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  • DOI: https://doi.org/10.1007/s10719-013-9499-1

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