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‡ 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
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.
Footnotes
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↵* This work was performed as part of a research and development project within the Industrial Science and Technology Frontier Program and was supported by the New Energy and Industrial Technology Development Organization.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF048687.
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↵§ To whom correspondence should be addressed: Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan. Tel.: 81-285-68-7459; Fax: 81-285-68-7452; E-mail: 387533{at}kastanet.kao.co.jp.
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↵1 The abbreviations used are: GSL, glycosphingolipids; GlcCer, glucosylceramide; LacCer, Galβ1–4GlcCer; LacCer synthase, UDP-galactose:glucosylceramide β-1,4-galactosyltransferase; GalT, galactosyltransferase; glycopeptide β-1,4-GalT, UDP-galactose:N-acetylglucosamine β-1,4-galactosyltransferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; DTT, dithiothreitol; WGA, wheat germ agglutinin; bp, base pair(s); kb, kilobase pair(s); GM2, GalNAcβ1–4(NeuAcα2–3)Galβ1–4GlcCer; GA2, GalNAcβ1–4Galβ1–4GlcCer; GA1, Galβ1–3GalNAcβ1–4Galβ1–4GlcCer; GM1, Galβ1–3GalNAcβ1–4(NeuAcα2–3)Galβ1–4GlcCer; GD1b, Galβ1–3GalNAcβ1–4(NeuAcα2–8NeuAcα2–3)Galβ1–4GlcCer.
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- Received February 9, 1998.
- Revision received March 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
