Genomic structure and promoter analysis of the human alpha1, 6-fucosyltransferase gene (FUT8).

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1,6-fucosyltransferase (alpha1,6FucT) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the asparagine-linked GlcNAc residue of complex N-glycans via alpha1,6-linkage. We have cloned the genomic DNA which encodes the human alpha1,6FucT gene ( FUT8 ) and analyzed its structure. It was found that the gene consists of at least nine exons spanning more than a 50 kbp genomic region, and the coding sequence is divided into eight exons. The translation initiation codon was located at exon 2, and thus exon 1 encodes only 5'-untranslated sequences. Transcription initiation site of FUT8 was determined by 5'-rapid amplification of the cDNA end and a primer-extension analysis using the total RNA isolated from SK-OV-3 cells, which have a high level of alpha1,6FucT activity. We then characterized the FUT8 promoter region by a reporter gene assay. The luciferase reporter assay indicated that the 5'-flanking region of exon 1, which covered about 1 kbp, conferred the promoter activity in SK-OV-3 cells. This region contains potential binding sites for some transcription factors, such as bHLH, cMyb, GATA-1, as well as a TATA-box, but not a CCAAT motif. 5'-Untranslated sequences found in ESTs and the cDNA for the FUT8 suggest the presence of an additional exon(s) at the upstream of the first exon identified in this study, and therefore, the transcription of the gene would be regulated by multiple promoters.

Mapping of the alpha-1,6-fucosyltransferase gene, FUT8, to human chromosome 14q24.3.

Alpha-1,6-Fucosyltransferase (alpha1,6FucT) is involved in the biosynthesis of asparagine-linked glycoprotein oligosaccharides. In this study, we isolated a genomic clone for the human alpha1,6FucT gene (FUT8) and mapped it by fluorescence in situ hybridization to chromosome 14q24.3. This study suggests a distinct localization of FUT8 from genes for other human fucosyltransferases reported to date.

Gene expression of alpha1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of alpha-fetoprotein.

The 1-6 fucosylated -fetoprotein (AFP) present in serum of patients with hepatocellular carcinoma (HCC) has been employed for the differential clinical diagnosis of HCC from chronic liver diseases. The molecular mechanism by which this alteration occurs, however, remains largely unknown. To address this issue, we purified GDP-L-Fuc:N-acetyl-beta-D-glucosaminide 1-6 fucosyltransferase (1-6 FucT), an enzyme involved in the 1-6 fucosylation of N-glycans from porcine brain, as well as from a human gastric cancer cell line, and cloned their genes. In this study, levels of 1-6 FucT mRNA expression and the activity of this enzyme for 12 human HCC tissues were examined and compared with that in surrounding tissues and normal livers. The mean +/- SD for 1-6 FucT activity was 78 +/- 41 pmol/h/mg in normal control liver, 202 +/- 127 pmol/h/mg in adjacent uninvolved liver tissues (chronic hepatitis: 181 +/- 106 pmol/h/mg; liver cirrhosis: 233 +/- 164 pmol/h/mg), and 195 +/- 72 pmol/h/mg in HCC tissues. The mRNA expression of 1-6 FucT was also enhanced in proportion to enzymatic activity except for a few cases, suggesting that 1-6 FucT expression is increased in chronic liver diseases, especially liver cirrhosis. Transfection of 1-6 FucT gene into cultured rat hepatocytes markedly increased 1-6 FucT activity and led to an increase in lens culinaris agglutinin (LCA) binding proteins in both cell lysates and condition media. When the 1-6 FucT gene was transfected into a human HCC cell line, Hep3B, which originally showed low levels of 1-6 FucT expression, 1-6-fucosylated AFP was dramatically increased in the condition media. Collectively, these results suggest that the enhancement of 1-6 FucT expression increased the fucosylation of several proteins, including AFP, and that the level of 1-6-fucosylated AFP in patients with HCC was in part caused by up-regulation of the 1-6 FucT gene expression.

Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT) from human gastric cancer MKN45 cells.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.

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.

Restriction endonuclease Aor13HI from Acidiphilium organovorum 13H, a new isoschizomer of BspMII: purification and characterization.

A restriction endonuclease, Aor13HI, an isoschizomer of BspMII, was purified to homogeneity from cell extracts of Acidiphilium organovorum strain 13H. The enzyme has a molecular mass of 60,000 daltons and consists of two subunits identical in molecular mass of 30,000 daltons. Aor13HI endonuclease, like BspMII, recognizes the palindromic six-base sequence 5'-TCCGGA-3', and cleaves between the T and C to produce a four-base 5' extension. Aor13HI is not inhibited by dam-dependent methylation. The isoelectric point of the enzyme is 5.7. Aor13HI activity was maximum at pH 7.5, 100 mM KCl, 7.5-10 mM MgCl2, and 55 degrees C. The enzyme was stable up to 60 degrees C. The N-terminal amino acid sequence (30 residues) of Aor13HI did not show any similarity with the sequence of other restriction endonucleases reported.