Expression Cloning of a Human Sulfotransferase That Directs the Synthesis of the HNK-1 Glycan on the Neural Cell Adhesion Molecule and Glycolipids

Abstract

The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcA␤133Gal␤134GlcNAc␤13 R, forming sulfo33GlcA␤133Gal␤134GlcNAc␤13 R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

“…This is compatible with the finding that GalNAc transferase catalyzing chondroitin chain elongation cannot transfer GalNAc to 3-O-sulfated GlcA (30). This sulfation may be accomplished either with a similar sulfotransferase, as reported for the detection of SO 4 -3GlcA␤1-4Xyl␤-4-methylumbelliferone from the substrate, 4-methylumbelliferyl ␤-Xyl (31), or with the sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope (32,33). These results suggest that glycosaminoglycan chain elongation of human TM seems to be abolished at the linkage tetrasaccharide core structure by addition of a sulfate group, indicating that 3-O-sulfation is a stop signal leading to TM lacking chondroitin sulfate.…”

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan‐protein linkage region of proteoglycans

“…Therefore, GlcAT-I seems to be mainly involved in the biosynthesis of the GAG-protein linkage region of proteoglycans. However, based on the present ¢ndings that the transfection of GlcAT-I cDNA into COS-1 cells induced a signi¢cant expression of the HNK-1 epitope, it is possible that tissues which abundantly express the GlcAT-I gene synthesize the HNK-1 epitope GlcA(3-O-sulfate)L1-3GalL1-4GlcNAc, especially since the HNK-1 3-O-sulfotransferase is widely distributed in various tissues and GlcAT-P most likely regulates the expression of the HNK-1 epitope [18,19]. In accordance with this suggestion, Wei et al recently reported the expression of the HNK-1 epitope in GlcAT-I cDNA-transfected COS-7 and Lec 2 cells when cotransfected with the HNK-1 3-O-sulfotransferase [14].…”

Molecular Cloning and Characterization of Chondroitin-4-O-sulfotransferase-3

“…The GalNAc-sulfotransferases add sulfate to the C4 hydroxyl of either terminal ␤1,4-linked GalNAc (GalNAc-4-ST1 (3,5) and GalNAc-4-ST2 (6)) or to ␤1,4-linked GalNAc that is substituted at its C-3 hydroxyl with either GlcUA (C4ST-1 (8, 9) and C4ST-3) or IdoUA (D4ST-1 (7)). In contrast, HNK-1 ST adds sulfate to the C-3 hydroxyl of terminal ␤1,3-linked GlcUA (1,2). Each member of this family of sulfotransferases is thus highly specific, and the unique sulfated structures that are produced have distinct biological roles.…”