Purification and characterization of a glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins from rat brain

Koji Terayama, Takashi Seiki, Akemi Nakamura, Kanae Matsumori, Satoru Ohta, Shogo Oka, Mutsumi Sugita, Toshisuke Kawasaki

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42 Citations (Scopus)

Abstract

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 90kDa 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 asialoneural 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.

Original languageEnglish
Pages (from-to)30295-30300
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number46
DOIs
Publication statusPublished - 13-11-1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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