Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synth ase (alpha 1,4-galactosyltransferase) was achieved using an anti-Gb3 antibo dy and mouse L cells as a recipient cell line for the transfection. The iso lated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane regi on, and a catalytic domain with 308 amino acids. Introduction of the cDNA c lone into L cells resulted in the neosynthesis of Gb3/CD77, and the extract s of the transfectant cells showed alpha 1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide, In Northern blotting, a 2 .3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and place nta and weakly in colon, small intestine, and brain. Transfection of the cD NA into L cells resulted in the constitution of sensitivity to the apoptosi s with Shiga-like toxins (verotoxins). Since Gh3/CD77 synthase initiates th e synthesis of globe series glycolipids, the isolation of this cDNA will ma ke possible further investigations into the function of its important serie s of glycolipids.