Formation of HNK-1 determinants and the glycosaminoglycan tetrasaccharide linkage region by UDP-GlcUA:galactose β1,3-glucuronosyltransferases

While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta 1,3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO(3)GlcUA beta 1, 3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Gal beta 1,3/4GlcNAc beta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Gal beta 1,3Gal beta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Gal beta 1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H,, Tone, Y,, Tamura, J,, Neumann, K. W,, Ogawa, T,, Oka, S,, Kawasaki, T,, and Sugahara, K, (1998) J. Biol. Chem. 273, 6615-6618), The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.