Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase from mung beans.
Substitution of the asparagine-linked GlcNAc by alpha1,3-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 alpha1,3-fucosyltransferase (core alpha1,3-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 alpha1,3-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 alpha1,3/4-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 GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4GlcNAcbeta1-Asn, GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4(Fucalpha1-6)GlcNAcbeta1-Asn, and GlcNAcbeta1-2Manalpha1-3[Manalpha1-3(Manalpha1-6 )Manalpha1-6]Manbeta1 -4GlcNAcbeta1-4GlcNAcbeta1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha1,3-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.
Leiter
H
H
Institute of Chemistry, Universität für Bodenkultur, Muthgasse 18, 1190 Wien, Austria. faltmann@edv2.boku.ac.at
Mucha
J
J
Staudacher
E
E
Grimm
R
R
Glössl
J
J
Altmann
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D005647:Fucosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D011134:Polysaccharides; D011994:Recombinant Proteins; D006154:Guanosine Diphosphate Fucose; D001216:Asparagine; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase