Purification, cDNA cloning, and expression of UDP-Gal: Glucosylceramide β-1,4-Galactosyltransferase from rat brain

Tomoko Nomura, Minoru Takizawa, Junken Aoki, Hiroyuki Arai, Keizo Inoue, Etsuji Wakisaka, Naonobu Yoshizuka, Genji Imokawa, Naoshi Dohmae, Koji Takio, Michihiro Hattori, Noboru Matsuo

Research output: Contribution to journalArticlepeer-review

119 Citations (Scopus)

Abstract

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N- glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse β-1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to β-1,4-GlcNAc in glycoproteins.

Original languageEnglish
Pages (from-to)13570-13577
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number22
DOIs
Publication statusPublished - 1998 May 29

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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