Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids

J Biol Chem. 1998 Feb 27;273(9):5190-5. doi: 10.1074/jbc.273.9.5190.

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, GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, forming sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Brain / enzymology
  • CHO Cells
  • Carbohydrate Sequence
  • Cell Adhesion / genetics
  • Cloning, Molecular
  • Cricetinae
  • DNA, Complementary / genetics
  • Epitopes / biosynthesis
  • Fetus / enzymology
  • Gene Expression
  • Glycolipids / biosynthesis*
  • Golgi Apparatus / enzymology
  • Humans
  • Molecular Sequence Data
  • Neural Cell Adhesion Molecules / biosynthesis*
  • Rats
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Sulfotransferases / genetics*
  • Sulfotransferases / metabolism
  • Tissue Distribution
  • Trisaccharides / biosynthesis*

Substances

  • DNA, Complementary
  • Epitopes
  • Glycolipids
  • Neural Cell Adhesion Molecules
  • Recombinant Proteins
  • Trisaccharides
  • CHST10 protein, human
  • Chst10 protein, rat
  • HNK-1 sulfotransferase
  • Sulfotransferases

Associated data

  • GENBANK/AF033827