Purification and characterization of a glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins from rat brain.
The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid, i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group.
Terayama
K
K
Department of Biological Chemistry and CREST (Core Research for Evolutional Science and Technology) Project, Japan Science and Technology Corporation, Kyoto, Japan.
Seiki
T
T
Nakamura
A
A
Matsumori
K
K
Ohta
S
S
Oka
S
S
Sugita
M
M
Kawasaki
T
T
D000818:Animals; D001921:Brain / Q000201:enzymology*; D018959:CD57 Antigens / Q000096:biosynthesis*; D002384:Catalysis; D002850:Chromatography, Gel; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D013109:Sphingomyelins / Q000378:metabolism; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000378:metabolism
D018959:CD57 Antigens; D006023:Glycoproteins; D013109:Sphingomyelins; D014530:Uridine Diphosphate; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase