Publications

• Product-identification and substrate-specificity studies of the GDP-L-fucose:2-acetamido-2-deoxy-beta-D-glucoside (FUC goes to Asn-linked GlcNAc) 6-alpha-L-fucosyltransferase in a Golgi-rich fraction from porcine liver.

  Longmore G.D., Schachter H.

  Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers L-fucose in alpha-(1 goes to 6) linkage from GDP-L-fucose to the asparagine linked 2-acetamido-2-deoxy-D-glucose residue of a glycopeptide derived from human alpha 1-acid glycoprotein. Product identificatio ... >> More

  Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers L-fucose in alpha-(1 goes to 6) linkage from GDP-L-fucose to the asparagine linked 2-acetamido-2-deoxy-D-glucose residue of a glycopeptide derived from human alpha 1-acid glycoprotein. Product identification was performed by high resolution, 1H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-L-fucose: 2-acetamido-2-deoxy-beta-D-glucoside (Fuc goes to Asn-linked GlcNAc) 6-alpha-L-fucosyltransferase, because the substrate requires a terminal beta-(1 goes to 2)-linked GlcNAc residue on the alpha-Man (1 goes to 3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contain 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal beta-(1 goes to 2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in beta-(1 goes to 4) linkage to the beta-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-alpha-L-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlCNAc residue that have been isolated to date do not contain a core L-fucosyl residue. << Less

  Carbohydr Res 100:365-392(1982) [PubMed] [EuropePMC]

• Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates.

  Paschinger K., Staudacher E., Stemmer U., Fabini G., Wilson I.B.

  Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis ... >> More

  Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate alpha1,6-fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-fucosyltransferases. On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts. << Less

  Glycobiology 15:463-474(2005) [PubMed] [EuropePMC]

• Crystal structure of mammalian alpha1,6-fucosyltransferase, FUT8.

  Ihara H., Ikeda Y., Toma S., Wang X., Suzuki T., Gu J., Miyoshi E., Tsukihara T., Honke K., Matsumoto A., Nakagawa A., Taniguchi N.

  Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also re ... >> More

  Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction. << Less

  Glycobiology 17:455-466(2007) [PubMed] [EuropePMC]

• Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-->6fucosyltransferase.

  Uozumi N., Yanagidani S., Miyoshi E., Ihara Y., Sakuma T., Gao C.-X., Teshima T., Fujii S., Shiba T., Taniguchi N.

  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 s ... >> More

  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. << Less

  J. Biol. Chem. 271:27810-27817(1996) [PubMed] [EuropePMC]

• A quantitative method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase activity with lectin affinity chromatography.

  Voynow J.A., Scanlin T.F., Glick M.C.

  A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosy ... >> More

  A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosylation of the asparagine-linked N-acetylglucosamine. The enzyme was extracted from human skin fibroblasts and incubated with GDP-[14C]fucose and a specific substrate, asialo-agalactotransferrin glycopeptide. The product of the enzyme reaction, [14C]fucose alpha 1----6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 M methyl alpha-D mannopyranoside. The method was shown to be specific after characterization of the lentil lectin-bound glycopeptides by enzyme degradation and affinity chromatography. Quantitation of the method was shown by several parameters, including the linearity of product formed with respect to time, GDP-[14C]fucose concentration and enzyme concentration. << Less

  Anal Biochem 168:367-373(1988) [PubMed] [EuropePMC]