Molecular Cloning and Characterization of a Human β-Gal-3′-sulfotransferase That Acts on Both Type 1 and Type 2 (Galβ1–3/1–4GlcNAc-R) Oligosaccharides

Abstract

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864 -4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5-and 3-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing ␤-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal␤1-3GalNAc␣-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO 3 -3Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-PA by two-dimensional 1 H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal␤1-3Glc-NAc-R) and type 2 (Gal␤1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel ␤-Gal-3-sulfotransferase gene family.Sulfated glycoconjugates, whose sulfate groups are biologically relevant, occur in a wide range of biological compounds (reviewed in Ref. 1), including glycoproteins, proteoglycans, glycolipids, and polysaccharides. The enzymes responsible for the sulfation of these compounds, sulfotransferases, utilize in common the sulfate donor 5Ј-phosphoadenosine 3Ј-phosphosulfate (PAPS). 1We recently reported on the purification of the glycolipid 3-sulfotransferase (cerebroside sulfotransferase (CST); galactosylceramide sulfotransferase, EC 2.8.2.11) to homogeneity from human renal cancer cells (2) and the cloning of the human CST cDNA on the basis of the amino acid sequence of the purified enzyme (3). The deduced amino acid sequence shows no overall homology to other sulfotransferases, except for the PAPS-binding motifs, suggesting that CST has a different evolutionary origin (3).Carbohydrate structures with 3Ј-sulfo-␤-Gal linkages have been found in both N-glycans (4, 5) and O-glycans (6 -13) of glycoproteins, and the ␤-Gal-3Ј-sulfotransferase activities responsible for the synthesis of these glycoproteins have been demonstrated (4, 13-16). The ␤-Gal-3Ј-sulfotransferase synthesizing O-glycans has bee...

Down‐regulation of Gal 3–O‐sulfotransferase‐2 (Gal3ST‐2) Expression in Human Colonic Non‐mucinous Adenocarcinoma

“…Interestingly, Gal3ST-1 also acts on β-Gal residues in pNP-or Bn-conjugated oligosaccharides, suggesting that Gal3ST-1 essentially recognizes hydrophobic moieties including Cer, pNP, and Bn, as well as β-Gal residues. Gal3ST-2 can act on type 1, type 2, and core 1 structure, as shown by Honke et al 10) It should be noted that the enzyme can utilize not only β-Gal residues, but also Galα-O-pNP, and that LNT can be sulfated only by Gal3ST-2 (Table IV). Gal3ST-3 prefers type 2 oligosaccharides to type 1 and core 1, as shown by El-Fasakhany et al 12) Gal3ST-4 is strictly core 1-specific, as shown by us.…”

“…Gal3ST transfers sulfate from PAPS to the C-3 position of Gal residues in various glycoconjugates. Four Gal3ST genes have so far been identified [9][10][11][12][13] and their genetic and biochemical characters are summarized in Table I. Honke et al 9,14) purified cerebroside 3′-sulfotransferase (Gal3ST-1) from human renal cancer cells and isolated its cDNA based on partial amino acid sequences.…”

“…Plasmids pSV-GP3ST, which is an expression vector encoding for Gal3ST-2, was a kind gift from Dr. K. Honke (Osaka University). 10) pCXNGalST encoding for Gal3ST-3 was a kind gift from Dr. T. Muramatsu (Nagoya University).…”

“…Gal3ST-2 has broad substrate specificity and is expressed in various tissues including colonic epithelial cells. 10) Gal3ST-3 exhibits rather limited expression. 11,12) Gal3ST-4 was identified by us and shown to have a characteristic strict substrate specificity for core 1 and core 2.…”

Sulphated galactosylceramide

“…In this context, some variants of ceramides are interesting too. In beta cells, ceramide is taken up and ligated with galactose by ceramide galactosyltransferase to β-galactosylceramide (GalCer) [3] and, furthermore in the Golgi apparatus, a sulphate group is linked by galactosylceramide sulfotransferase to form sulphated galactosylceramide, also called sulfatide [4]. The same process takes place in the brain and is likely to occur in the liver.…”