cDNA CLONING OF HUMAN ALPHA-1-6 FUCOSYLTRANSFERASE AND ITS APPLICATION IN THE EARLY DIAGNOSIS OF HEPATOCELLULAR CARCINOMA.

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Early diagnosis of hepatocellular carcinoma (HCC)is very important during the follow-up of patients with chronic liver diseases. We have previously reported that the measurement of Lens culinaris agglutinin (LCA) -reactive species of alpha-fetoprotein, alpha-1-antitrypsin and transferrin is a useful marker for the early detection of HCC.The molecular basis for the affinity for LCA is the fucosylation at the innermost N-acetylglucosamine residue in the biantennary carbohydrate chain. However, precise enzymatic background of this core (alpha 1-6) fucosylation has not been fully understood in human HCC.In the present project we tried to perform the measurement of alpha 1-6 fucosyltransferase (AFT) activity and its purification and cDNA cloning. AFT activity was determined by the procedure as follows : First, oligosaccharides were obtained from human serum transferrin by pronase digestion with subsequent gelfiltration by Toyopearl HW-40S (3.0 * 90 cm) equilibrated with 1 M acetic acid. Nex … More t, fluorescence labeling of oligosaccharides was performed with pyridylamino-ethyl-succinamic acid 5-norborenedicarboximide. The oligosaccharide fraction was purified by HPLC on a Amide-80, and lyophilized. The standard reaction mixture is 125 mM 2- (N-Morpholino) ethanesulfonic acid-NaOH buffer of pH 7.0 containing 30 mu M of substrate, 500 mu M of GDP-fucose, 20 mM of N-acetylglucosamine, 1% Triton X-100 and sample, in a final volume of 50 mu l. After incubation at 37ﾟC for 5 h, the reaction was stopped by the addition of 10 mu 1 of 2% sodium-tetraborate-0.25 M EDTA.Then the product was applied to high performance liquid chromatography (Hitachi 6200A) equipped with a Rheodyne Model 7125 injector and a Hitachi Model F-1050 fluorescence spectrophotometer on a reversed-phase column (PALPAK type R-MB,0.21 * 15 cm, Takara Shuzo Co., Ltd.Kyoto, Japan). Elution was performed at a flow rate of 0.5 ml/min at 40ﾟC using two solvents, A and B.Solvent A was 0.1 M acetic acid triethylamine buffer of pH 4.0 and solvent B was solvent A with 0.5% 1-butanol. The column was equilibrated with a mixture of solvent A (95%) and B (5%). After injection of a sample, the ratio of solvent B to A was increased linearly to 55% solvent B in 80 min. Pyridylamino (PA) -oligosaccharides were detected by fluorescence using excitation and emission wavelengths of 320 and 400 nm, respectively. When we finished to develop the measurement of AFT activity and purification of AFT,Taniguchi N and his coworker reported cDNA cloning of this enzyme from porcine brain. According to their report, porcine brain AFT has 575 amino acid and belonged to type II transmembrane protein like other glycosyltransferase. Therefore, 2 oligosaccharides were synthesized for use as primer in PCR according to the cDNA sequence of catalytic domain in the carboxy-terminal region of porcine AFT.Reproducible PCR products (mRNA) were obtained from Hep G2. Accordingly, the PCR products are going to be subcloned into a vector for cD Less