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

人α-1-6岩藻糖基转移酶的cDNA克隆及其在肝癌早期诊断中的应用。

• 批准号: 07457132
• 负责人: AOYAGI Yutaka
• 金额: $ 3.78万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457132/
• 关键词: alpha 1-6 fucosyltransferase; fucosylation; hepatocellular carcinoma; cDNA cloning; Lens culinaris agglutinin; early diagnosis

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

在慢性肝病患者的随访中，肝细胞癌的早期诊断是非常重要的。我们先前已经报道，对晶状体凝集素(LCA)活性物种甲胎蛋白、α-1-抗胰蛋白酶和转铁蛋白的测定是早期检测肝癌的有用标志。与LCA亲和力的分子基础是双触角碳水化合物链最内侧N-乙酰氨基葡萄糖残基的岩藻糖化。然而，这一核心(α1-6)岩藻糖基化的确切的酶背景在人肝癌中还不完全清楚。在本项目中，我们试图对α1-6岩藻糖基转移酶(AFT)的活性进行测定，并对其进行纯化和克隆。测定AFT活性的方法如下：首先，从人血清转铁蛋白中提取低聚糖，用链霉蛋白酶消化，然后用Toyota opearl HW-40S(3.0×90 cm)凝胶过滤，然后用1M的冰醋酸平衡。NEX…用吡啶氨乙基丁二酰亚胺5-降冰片二酰亚胺对寡糖进行荧光标记。低聚糖组分在酰胺-80柱上用高效液相色谱分离纯化，并冷冻干燥。标准反应混合物为125mM2-(N-吗啉)乙磺酸-氢氧化钠缓冲液，其中含有底物30mM，岩藻糖500mM，N-乙酰氨基葡萄糖20 mM，1%Triton X-100和样品，最后体积为50亩L。在37ﾟC下孵育5h，在反相柱(PALPAK型R-MB，0.21×15 cm，日本京都Takara Shuzo Co.，0.21×15 cm)上用Rheodyne 7125型进样器和日立F-1050型荧光分光光度计进行高效液相色谱(Hitachi 6200A)。洗脱剂A为0.1M醋酸三乙胺缓冲液(pH 4.0)，B为含0.5%正丁醇的溶剂A，洗脱温度40ﾟC，流速0.5ml/m in。用A(95%)和B(5%)混合溶剂平衡色谱柱。进样后，溶剂B与A的比例在80分钟内线性增加到55%B。以320 nm为激发波长，400 nm为发射波长，用荧光法测定了吡啶氨基低聚糖的含量。当我们完成了AFT活性的测定和AFT的纯化工作后，Taniguchi N和他的同事报道了从猪脑中克隆出这种酶的cDNA。根据他们的报道，猪脑AFT含有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个寡糖用于聚合酶链式反应。从Hep G2中获得了重复性较好的扩增产物。因此，聚合酶链式反应产物将被亚克隆到CD较少的载体中

项目成果

青柳 豊: "Alpha-fetoprotein producing renal cell carcinoma with the increased activity of N-acetylglucosaminyltransferase III." Nephron,. 74. 409-414 (1996)

Yutaka Aoyagi：“N-乙酰氨基葡萄糖转移酶 III 活性增加的产生甲胎蛋白的肾细胞癌”，74. 409-414 (1996)。

Hirota S.Nomoto, M.Hosobe, S.Aoyagi, Y.and Asakura, H.: "Mucin in primary liver carcinoma : combined hepatocellular-cholangiocarcinoma or variant hepatocellular carcinoma" The Canadian J Gastroenterology. 10. 12-16 (1996)

Hirota S.Nomoto、M.Hosobe、S.Aoyagi, Y. 和 Asakura, H.：“原发性肝癌中的粘蛋白：联合肝细胞胆管癌或变异型肝细胞癌”《加拿大胃肠病学杂志》。

Tsuchida, K.Aoyagi, Y.Odani, S.Mita, T.and Isemura, M.: "Isolation of a nobel collagen-binding protein from the mushroom, Hypsizigus marmoreus which inhibits the Lewis lung carcinoma cell adhesion to type IV collagen." J Biol Chem. 270. 1481-1484 (1995)

Tsuchida, K.Aoyagi, Y.Odani, S.Mita, T. 和 Isemura, M.：“从蘑菇 Hypsizigus marmoreus 中分离出一种诺贝尔胶原蛋白结合蛋白，该蛋白可抑制 Lewis 肺癌细胞与 IV 型胶原蛋白的粘附。

土田和孝: "Isolation of nobel collagen-binding protein from the mushroom,Hypsizigus marmoreus which inhibits the Lewis lung carcinoma cell adhesion to type IV collagen." J Biol Chem. 270. 1481-1484 (1995)

Kazutaka Tsuchida：“从蘑菇中分离出诺贝尔胶原蛋白结合蛋白，抑制 Lewis 肺癌细胞与 IV 型胶原蛋白的粘附。” J Biol Chem. 270. 1481-1484 (1995)

青柳豊: "消化器病Up to date,Consensus and Controversies,1996" 編集代表:小林絢三,永井書店, 396 (1997)

Yutaka Aoyagi：“胃肠道疾病最新、共识和争议，1996” 总编辑：Kenzo Kobayashi，Nagai Shoten，396（1997）