Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.
We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.
Ichikawa
S
S
Laboratory for Glyco-Cell Biology, Frontier Research Program, The Institute of Chemical and Physical Research, Saitama, Japan.
Sakiyama
H
H
Suzuki
G
G
Hidari
K I
KI
Hirabayashi
Y
Y
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D051146:N-Acylsphingosine Galactosyltransferase; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution
D018076:DNA, Complementary; D006028:Glycosphingolipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D051146:N-Acylsphingosine Galactosyltransferase; C023246:ceramide glucosyltransferase