Expression Cloning of a cDNA for Human Ceramide Glucosyltransferase that Catalyzes the First Glycosylation Step of Glycosphingolipid Synthesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 10; pp. 4638 - 4643
• Main Authors: Ichikawa, Shinichi, Sakiyama, Hisako, Suzuki, Gen, Hidari, Kazuya I.-P. Jwa, Hirabayashi, Yoshio
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 14.05.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; Amino acids; Base Composition; Base Sequence; Biochemistry; Cell Line; Cell lines; Ceramides; Cloning, Molecular; Complementary DNA; Deoxyribonucleic acid; DNA; DNA, Complementary - chemistry; DNA, Complementary - genetics; Enzymes; Escherichia coli - genetics; Galactosyltransferases - genetics; Gene Expression; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glycosphingolipids - biosynthesis; Glycosylation; Humans; Ice; Lipids; Membranes; Messenger RNA; Molecular Sequence Data; Mutation; N-Acylsphingosine Galactosyltransferase; Open Reading Frames; Plasmids; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Skin cancer; Tissue Distribution
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.10.4638

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 hydorphobicity was detected in the region 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 evolutionalary origins of these enzymes.