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J Biol Chem, Vol. 274, Issue 31, 21830-21839, July 30, 1999

Purification, cDNA Cloning, and Expression of GDP-L-Fuc:Asn-linked GlcNAc 1,3-Fucosyltransferase from Mung Beans

Haralt Leiter, Jan Mucha, Erika Staudacher, Rudolf Grimm^§, Josef Glössl, and Friedrich Altmann

From the Institute of Chemistry, the  Centre of Applied Genetics, Universität für Bodenkultur, Muthgasse 18, 1190 Wien, Austria and ^§ Hewlett-Packard, Waldbronn, Germany

Substitution of the asparagine-linked GlcNAc by 1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-L-Fuc:Asn-linked GlcNAc 1,3-fucosyltransferase (core 1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core 1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial 1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc1-Asn, GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc1-Asn, and GlcNAc1-2Man1-3[Man1-3(Man1-6)Man1-6]Man1-4GlcNAc1-4GlcNAc1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core 1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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