Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis.
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-[(3)H]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.
Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.
Tadahiro
T
Kumagai
Takeshi
T
Sato
Shunji
S
Natsuka
Yukito
Y
Kobayashi
Dapeng
D
Zhou
Tadashi
T
Shinkai
Satoru
S
Hayakawa
Kiyoshi
K
Furukawa
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis*; D002460:Cell Line; D049109:Cell Proliferation; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D006017:Glycolipids / Q000378:metabolism; D006031:Glycosylation; D007790:Lactosylceramides / Q000096:biosynthesis*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D013379:Substrate Specificity
D015703:Antigens, CD; D006017:Glycolipids; D007790:Lactosylceramides; D008562:Membrane Glycoproteins; C009744:CDw17 antigen; D005700:Galactosyltransferases; C419408:beta-1,4-galactosyltransferase V