Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-->6fucosyltransferase.
GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-->6fucosyltransferase (alpha1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 2)Manbeta1-4GlcNAcbet a1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440, 000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The alpha1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. alpha1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.
Uozumi
N
N
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.
Yanagidani
S
S
Miyoshi
E
E
Ihara
Y
Y
Sakuma
T
T
Gao
C X
CX
Teshima
T
T
Fujii
S
S
Shiba
T
T
Taniguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014162:Transfection
D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase