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

在慢性肝病患者的随访中，早期诊断肝细胞癌是非常重要的。我们之前报道过，测定鸡眼凝集素(LCA) -甲胎蛋白，α -1-抗胰蛋白酶和转铁蛋白的反应种是早期检测HCC的有用标志物。LCA亲和的分子基础是双触角碳水化合物链中最内层n -乙酰氨基葡萄糖残基的聚焦。然而，在人类HCC中，这种核心（α 1-6）聚焦化的确切酶学背景尚未完全了解。在本项目中，我们试图进行α 1-6聚焦转移酶（AFT）活性的测定及其纯化和cDNA克隆。测定AFT活性的方法如下：首先，用pronase酶切法从人血清转铁蛋白中获得低聚糖，然后用Toyopearl HW-40S （3.0 * 90 cm）凝胶过滤，并用1 M醋酸平衡。然后，用吡啶氨基-乙基琥珀酸5-去甲硼二酰亚胺对低聚糖进行荧光标记。在Amide-80上高效液相色谱纯化低聚糖部分，并进行冻干。标准反应混合物为125 mM 2- (N-Morpholino)乙基磺酸- naoh缓冲液，pH 7.0，含底物30 μ M， GDP-浓缩液500 μ M， n -乙酰氨基葡萄糖20 mM， 1% Triton X-100和样品，终体积为50 μ l。37℃孵育5 h后，加入10 μ 1 2%四硼酸钠-0.25 M EDTA停止反应。然后使用Rheodyne型号7125进样器和日立型号F-1050荧光分光光度计，在反相柱（PALPAK型R-MB，0.21 * 15 cm, Takara Shuzo Co., Ltd.Kyoto, Japan）上进行高效液相色谱（Hitachi 6200A）。洗脱液为a、B两种溶剂，流速为0.5 ml/min，洗脱温度为40ºC，溶剂a为0.1 M pH为4.0的乙酸三乙胺缓冲液，溶剂B为含0.5% 1-丁醇的溶剂a。用溶剂a（95%）和B（5%）的混合物平衡柱。样品进样后，溶剂B与a的比例在80分钟内线性增加至溶剂B的55%。利用激发波长为320 nm、发射波长为400 nm的荧光法检测Pyridylamino (PA) -寡糖。当我们完成对AFT活性的测定和AFT的纯化时，Taniguchi N和他的同事报道了从猪脑中克隆该酶的cDNA。根据他们的报告，猪脑AFT有575个氨基酸，与其他糖基转移酶一样属于II型跨膜蛋白。因此，根据猪aft羧基末端催化结构域的cDNA序列，合成了2个低聚糖作为引物，获得了可重复的PCR产物（mRNA）。因此，PCR产物将被亚克隆到cD Less的载体中

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