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 (g lucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltran sferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylatio n step of glycosphingolipid synthesis and the product, glucosylceramid e, serves as the core of more than 300 glycosphingolipids. The cDNA ha s a G+C-rich 5' untranslated region of 290 nucleotides and the open re ading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequ ence. In addition, considerable hydrophobicity was detected in the reg ions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfe cted with the cDNA, Northern blot analysis revealed a single transcrip t of 3.5 kb, and the mRNA was widely expressed in organs. The amino ac id sequence of ceramide glucosyltransferase shows no significant homol ogy to ceramide galactosyltransferase, which indicates different evolu tionalary origins of these enzymes.