Molecular Cloning of Globotriaosylceramide/CD77 Synthase, a Glycosyltransferase That Initiates the Synthesis of Globo Series Glycosphingolipids

• Published in: The Journal of biological chemistry Vol. 275; no. 20; pp. 15152 - 15156
• Main Authors: Kojima, Yoshinao, Fukumoto, Satoshi, Furukawa, Keiko, Okajima, Tetsuya, Wiels, Joelle, Yokoyama, Keiko, Suzuki, Yasuo, Urano, Takeshi, Ohta, Michio, Furukawa, Koichi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.05.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Catalytic Domain; Cloning, Molecular; Gene Library; globotriaosylceramide/CD77 synthase; glycolipids; Glycosphingolipids - biosynthesis; Humans; Kinetics; L Cells; Melanoma; Mice; Molecular Sequence Data; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Transfection; Trihexosylceramides - chemistry; Trihexosylceramides - genetics; Trihexosylceramides - metabolism; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M909620199

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (α1,4-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 α1,4-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.