Purification and cDNA Cloning of Porcine Brain GDP-L-Fuc:N-Acetyl-β-D-Glucosaminide α1→6Fucosyltransferase

Abstract

GDP-L-Fuc:N-acetyl-␤-D-glucosaminide ␣136fucosyl-transferase (␣1-6FucT; 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 GlcNAc␤1-2Man␣1-6(GlcNAc␤1-2Man␣1-2)-Man␤1-4GlcNAc␤1-4GlcNAc-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 ␣1-6FucT 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. ␣1-6FucT activity was found to be high in the transfected cells, as compared with non-or mocktransfected cells. Northern blotting analyses of rat adult tissues showed that ␣1-6FucT 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.It has been reported that the structures of glycopeptides change during the development and differentiation of embryos (1-4). Detailed analysis of specific antigens on the surface of various carcinoma cells revealed that carcinoma-specific sugar chains are expressed on the cell surface. A well documented phenotypic alteration of these specific sugar chains is the increase in the molecular weight of cell surface complex type N-linked glycan in transformed cells. This change has been observed regardless of the nature of the transforming agent: oncogenic viruses (5-9), chemical mutagens (10 -11), or DNA from unrelated tumor cells (12)(13)(14). This phenomenon was thought to reflect the deviation of carcinoma cells from the ordinary differentiation processes. ␣-Fucose residue attached to asparagine-linked GlcNAc also have some relationship with carcinogenesis. A difference in the binding pattern of serum ␣-fetoprotein with lentil lectin between hepatocellular carcinomas and benign liver diseases has been reported (15-17). Analyses of the carbohydrate structure of ␣-fetoprotein from hepatocellular carcinoma cell lines have indicated that almost all of the carbohydrates of ␣-fetoprotein are ␣1-6-fucosylated (18). ␣-Fetoprotein produced by germ cell tumors, such as yolk sac tumors, is also highly fucosylated (19). The activity of ␣1-6FucT 1 was higher in hepatocellular carcinoma tissue than in non-tumor tissue (20) and was induced by the transfection of the ras protooncogene into 3T3 fibroblast cells (21). Schachter et al. (22,23) first characterized ␣1-6FucT in porcine liver using a partially purified enzyme extract. The special release of ␣1-6FucT from platelets during blood clotting has been reported (24, 25), alteration of fucosylation has been reported in cystic fibrosis glycoproteins from different s...

A Novel Core Fucose-specific Lectin from the Mushroom Pholiota squarrosa

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Fucosylation of N-Glycans Regulates the Secretion of Hepatic Glycoproteins into Bile Ducts

“…Fucosylated ␣-fetoprotein (AFP) 2 , referred to as the L3 band of AFP on LCA (Lensculinaris agglutinin) lectin affino electrophoresis, is a highly specific tumor marker for hepatocellular carcinoma (HCC). Increases in AFP levels in serum are observed in patients with chronic liver disease such as liver cirrhosis, but fucosylated AFP is barely detectable in benign liver diseases (1).…”

Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics

“…The enzyme was first identified and reported by Schachter' group in 1980 (10). Glick's group (84) subsequently attempted to purify the enzyme but our group finally successfully purified it from a human cancer cell line as well as porcine brain tissues by affinity chromatography using core fucosylated biannetennary sugar chain as a solid ligand (85,86). KO mice were successfully produced by our group and we found that most Fut8 KO mice die within 3 days and the survivors show growth retardation and finally eventually develop emphysematous changes of the lung at 3 weeks after birth (87).…”