Molecular Cloning and Characterization of a Novel Human Galactose 3-O-Sulfotransferase That Transfers Sulfate to Galβ1→3GalNAc Residue in O-Glycans

Abstract

We have identified a novel galactose 3-O-sulfotransferase, termed Gal3ST-4, by analysis of an expression sequence tag using the amino acid sequence of human cerebroside 3-sulfotransferase (Gal3ST-1). The isolated cDNA contains a single open reading frame coding for a protein of 486 amino acids with a type II transmembrane topology. The amino acid sequence of Gal3ST-4 revealed 33%, 39%, and 30% identity to human Gal3ST-1, Gal␤133/ 4GlcNAc:33-sulfotransferase (Gal3ST-2) and Gal␤13 4GlcNAc:33-sulfotransferase (Gal3ST-3), respectively. The Gal3ST-4 gene comprised at least four exons and was located on human chromosome 7q22. Expression of Gal3ST-4 in COS-7 cells produced a sulfotransferase activity that catalyzes the transfer of [ 35 S]sulfate to the C-3 position of Gal␤133GalNAc␣1-O-Bn. Gal3ST-4 recognizes Gal␤133GalNAc and Gal␤133 (GlcNAc␤136)GalNAc as good substrates, but not Gal␤133GalNAc OH or Gal␤133/4GlcNAc. Asialofetuin is also a good substrate, and the sulfation was found exclusively in O-linked glycans that consist of the Gal␤133GalNAc moiety, suggesting that the enzyme is specific for O-linked glycans. Northern blot analysis revealed that 2.5-kilobase mRNA for the enzyme is expressed extensively in various tissues. These results suggest that Gal3ST-4 is the fourth member of a Gal:33-sulfotransferase family and that the four members, Gal3ST-1, Gal3ST-2, Gal3ST-3, and Gal3ST-4, are responsible for sulfation of different acceptor substrates.

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

“…13) Briefly, the labeled oligosaccharides were dissolved in 20 µl of 75 mM sodium metaperiodate, 75 mM sodium acetate (pH 5.3) and incubated at 4°C for 24 h in the dark. After destruction of excess periodate, 300 µl of 0.1 M sodium borate (pH 9.0) containing 0.1 M sodium borohydride was added, and the solution stood for 1 h at room temperature.…”

“…13) cDNA cloning of Gal3ST-1 The cDNA encoding for Gal3ST-1 9) was amplified from "Super Script" human testis cDNA library (Life Technologies, Inc., Rockville, MD) by PCR. Oligonucleotide primers used for the PCR were 5′-tttaagcttGTCTGAGATGCTGCCA-3′ (forward primer) and 5′-tttggatccTGGGACGTCACCACCG-3′ (reverse primer).…”

“…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.…”

Evidence for GAL3ST4 mutation as the potential cause of pectus excavatum

“…Gal3ST-4 is the fourth member of the Gal:→3-sulfotransferase family that catalyzes the C-3 sulfation of galactoses in O-linked glycoproteins [5]. It has been shown that sulfation of the glycan chains is associated with the specific physiological functions of the various glycoproteins.…”

“…Thus, it can be conceived that dysfunction of GAL3ST4 by genetic mutations will have a great effect on the sulfation of the various target glycoproteins. The four members of the Gal:→3-sulfotransferase family share 30%-40% amino acid sequence identity, but each of them shows distinct substrate specificities [5]. Although it has been shown that the Gal3ST-4 is highly specific for the core 1 structures generated in the O-linked glycans [10], future studies are needed to unravel the exact downstream targets or pathways of Gal3ST-4 that contribute to the development of familial PE.…”