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Glycoconjugate Journal

, Volume 27, Issue 7–9, pp 685–695 | Cite as

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

  • Tadahiro Kumagai
  • Takeshi Sato
  • Shunji Natsuka
  • Yukito Kobayashi
  • Dapeng Zhou
  • Tadashi Shinkai
  • Satoru Hayakawa
  • Kiyoshi FurukawaEmail author
Article

Abstract

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5 +/− - and B4galt5 −/− -derived MEF cells are a half and null when compared to that of B4galt5 +/+ -derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5 +/+ -, B4galt5 +/− - and B4galt5 −/− -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 +/+ -, B4galt5 +/− - and B4galt5 −/− -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[3H]Gal, Lac-Cer synthase activity in B4galt5 +/− - and B4galt5 −/− -derived MEF cells decreased to 41% and 11% of that of B4galt5 +/+ -derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 −/− -derived MEF cells decreased remarkably when compared with those of B4galt5 +/+ -derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.

Keywords

B4galt5−/− mice MEF cells β-1,4-GalT V Lactosylceramide 

Abbreviations

β-1,4-GalT

β-1,4-galactosyltransferase

B4galt5

β-1,4-GalT V gene

CBB

Coomassie Brilliant Blue

Con A

Concanavalin A

Gal

Galactose

Glc-Cer

glucosylceramide

GlcNAcβ-S-pNP

p-nitrophenyl-N-acetyl-1-thio-β-D-glucosaminide

GU

Glucose unit

GSL

Glycosphingolipid

HPLC

High performance liquid chromatography

Lac-Cer

Lactosylceramide

MAA

Maackia amurensis agglutinin

MEF

Mouse embryonic fibroblast

MES

2-(N-morpholino)ethansulfonic acid

Neu5Ac

N-acetylneuraminic acid

PA

Pyridylamine

PBS

10 mM phosphate-buffered saline (pH 7.4)

PNA

Peanut agglutinin

RCA-I

Ricinus communis agglutinin-I

RT-PCR

Reverse transcription-polymerase chain reaction

SNA

Sambucus nigra agglutinin

TLC

Thin-layer chromatography

Notes

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research (09240104, 12680708 and 22370048) from the Ministry of Education, Science, Culture and Sports of Japan, and the Research Promotion Fund from the Japan Science Technology Agency (2007-2009) to KF.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tadahiro Kumagai
    • 1
  • Takeshi Sato
    • 1
  • Shunji Natsuka
    • 2
  • Yukito Kobayashi
    • 1
  • Dapeng Zhou
    • 3
  • Tadashi Shinkai
    • 4
  • Satoru Hayakawa
    • 4
  • Kiyoshi Furukawa
    • 1
    Email author
  1. 1.Laboratory of Glycobiology, Department of BioengineeringNagaoka University of TechnologyNiigataJapan
  2. 2.Department of Biology, Faculty of ScienceNiigata UniversityNiigataJapan
  3. 3.Department of Melanoma Medical OncologyUniversity of Texas MD Anderson Cancer CenterHoustonUSA
  4. 4.Department of Cell BiologyTokyo Metropolitan Institute of GerontologyTokyoJapan

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