EXPRESSION CLONING OF A HUMAN SULFOTRANSFERASE THAT DIRECTS THE SYNTHESIS OF THE HNK-1 GLYCAN ON THE NEURAL CELL-ADHESION MOLECULE AND GLYCOLIPIDS

The HNK-1 carbohydrate is expressed on various adhesion molecules in t he nervous system and is suggested to play a role in cell-cell and cel l-substratum interactions, Here we describe the isolation and function al expression of a cDNA encoding a human sulfotransferase that synthes izes the HNK-1 carbohydrate epitope, A mutant Chinese hamster ovary ce ll line, Lec2, which stably expresses human neural cell adhesion molec ule (N-CAM) (Lec2-NCAM), was first established, Lec2-NCAM was co trans fected 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 L ec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescen ce-activated cell sorting, Sibling selection of recovered plasmids res ulted 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 me mbrane protein, Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homol ogous to the corresponding sequence in other Gels-associated sulfotran sferases so far cloned, The amount of HNK-1ST transcript is high in fe tal brain compared with fetal lung, kidney, and liver, Expression of H NK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a s oluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcA beta 1-->3Gal beta 1-->4GlcNAc beta 1-->R, forming sul fo-->3GlcA beta 1-->3Gal beta 1-->4GlcNAc beta 1-->R. The results comb ined together indicate that the cloned HNK-1ST directs the synthesis o f the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.