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Gewählte Publikation:

Leiter, H; Mucha, J; Staudacher, E; Grimm, R; Glössl, J; Altmann, F; .
(1999): Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase from mung beans.
J Biol Chem. 1999; 274(31):21830-21839 FullText FullText_BOKU

Abstract:
Substitution of the asparagine-linked GlcNAc by alpha 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 alpha 1,3-fucosyltransferase (core alpha 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 alpha 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 alpha 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 GlcNAc beta 1-2Man alpha 1-3(GlcNAc beta 1-2Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc beta 1-Asn, GlcNAc beta 1-2Man alpha 1-3(GlcNAc beta 1-2Man alpha 1-6)Man beta-1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc beta 1-Asn, and GlcNAc beta-1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4-GlcNAc beta 1-4GlcNAc beta 1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha 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.
Autor*innen der BOKU Wien:
Altmann Friedrich
Glößl Josef
Staudacher Erika
Find related publications in this database (using NML MeSH Indexing)
Amino Acid Sequence;Animals;Asparagine;Base Sequence;Carbohydrate Sequence;Chromatography, Affinity;Chromatography, DEAE-Cellulose;Chromatography, Ion Exchange;Cloning, Molecular;DNA, Complementary;Fabaceae/enzymology*;Fabaceae/genetics;Fucosyltransferases/genetics;Fucosyltransferases/isolation & purification*;Fucosyltransferases/metabolism*;Guanosine Diphosphate Fucose/metabolism;Humans;Introns;Molecular Sequence Data;Open Reading Frames;Plants, Medicinal*;Polysaccharides/chemistry;Polysaccharides/metabolism;Recombinant Proteins/isolation & purification;Recombinant Proteins/metabolism;Reverse Transcriptase Polymerase Chain Reaction;Sequence Alignment;Sequence Homology, Amino Acid;Substrate Specificity;



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