Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.

We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.