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

Abstract

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. 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 ␣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 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.

Molecular Cloning of Pigeon UDP-galactose:β-d-Galactoside α1,4-Galactosyltransferase and UDP-galactose:β-d-Galactoside β1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Galα1–4Galβ1–4Galβ1–4GlcNAc Sequence

“…There are three sequences in zebra finch and one sequence in chicken, and these sequences were annotated "similar to ␣1,4-galactosyltransferase." The BLAST analysis also revealed human (16,17), mouse (18), and rat (19) ␣4GalT(Gal)s as being homologous proteins, as well as some other predicted proteins from mammals that were annotated similar to ␣1,4-galactosyltransferase. The pigeon ␣4GalT(Gal) also has some homology with the human ␣1,4-N-acetylglucosaminyltransferase (␣1,4-N-acetylglucosaminyltransferase, 36.3% identity; accession number AAD48406) and putative ␣1,4-N-acetylglucosaminyltransferase from chicken (34.4%, accession number XP_426692) and zebra finch (31.5%, accession number XP_002189476).…”

“…Because the Gal␣1-4Gal sequence is rarely attached to glycoproteins in mammals, the putative ␣4GalT(Gal) in pigeon is likely to have distinct acceptor substrate specificities from those of mammals. Although mammalian ␣4GalT(Gal)s, also known as Gb3 synthases, have been cloned from human (16,17), mouse (18), and rat (19), they utilize lactosylceramide (Gal␤1-4Glc-Cer) as the preferred acceptor substrate to produce Gb3. Only when the expression level of the enzyme is sufficiently high, human Gb3 synthase can also synthesize P 1 antigen using lacto-N-neotetraosylceramide (Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-Cer) as a substrate (20).…”

The Blood Group P1 Synthase Gene Is Identical to the Gb3/CD77 Synthase Gene

“…On the other hand, the occurrence of the p phenotype is very rare in all nationalities (18,19), suggesting that the genetic basis of p and P2 phenotypes is not directly linked. Actually, no relevant polymorphic sequences in the coding region of the Gb3/CD77 synthase gene could be detected between P1 and P2 individuals, and no P1 synthase activity could be found in cell lysates transfected with a Gb3/CD77 synthase expression vector (8,9). Consequently, it appears difficult to accept that P1 synthase and Gb3/CD77 synthase are identical.…”

“…Enzyme Assay-Gb3/CD77 synthase activity was measured as described previously (8). The enzyme activity of ␣1,4Gal-T to generate P1 structure was measured as described previously (26).…”

“…The enzyme activity of ␣1,4Gal-T to generate P1 structure was measured as described previously (26). Briefly, membrane fractions were prepared as described (8). The reaction mixture for the assay contained the following in a volume of 50 l: 0.2 mM UDP-Gal (Sigma), UDP-[ 14 C]Gal (2.5 ϫ 10 5 dpm) (PerkinElmer Life Sciences), 2.5 g of nLc4, 20 M CDP-choline, and 100 g of phosphatidylglycerol (Sigma), and after evaporation, 20 mM sodium cacodylate-HCl, pH 6.8, 10 mM MnCl 2 , 20 mM galactonolactone (Sigma), 0.3% Triton X-100 (Sigma), 250 g of ␣-lactalbumin, and membrane fraction containing 100 g of protein.…”