Abstract
The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.
Highlights
The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (␣1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection
Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed ␣1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide
Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids
Summary
The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (␣1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed ␣1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. The isolated cDNA revealed that Gb3/CD77 synthase is a type II membrane protein with no significant homology with any other galactosyltransferase genes cloned so far.
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