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J Biol Chem, Vol. 273, Issue 22, 13570-13577, May 29, 1998

Purification, cDNA Cloning, and Expression of UDP-Gal: Glucosylceramide -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, and Noboru Matsuo

From the  Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan, the  Division of Biomolecular Characterization, Institute of Physical and Chemical Research (RIKEN), 2-1, Hirosawa, Wako, Saitama 351-0198, Japan, and the ^¶ Faculty of Pharmaceutical Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan

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 -1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to -1,4-GlcNAc in glycoproteins.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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