Decaens C, Nardelli J, Bara J, Burtin P
CNRS URA 1343, Institut Curie, Bat. 110, Centre Universitaire, Orsay, France.
Biochem J. 1993 Jul 15;293 ( Pt 2)(Pt 2):531-6. doi: 10.1042/bj2930531.
The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.
660表位是由一种针对大鼠胃表面上皮刮片产生的单克隆抗体所定义的。这个表位是杯状细胞分化的标志物，在结肠黏膜中表现出癌胚行为。我们发现它与胃粘蛋白糖蛋白共纯化。我们使用标准技术分离大鼠胃粘液糖蛋白：将PBS中的胃刮片在存在蛋白酶抑制剂的情况下于CsCl中进行等密度梯度离心。相对密度为1.4 - 1.45且中性糖/蛋白比率高的级分在Ultrogel A4柱上进行色谱分析。根据通常的标准，在排除体积中回收的高分子量糖蛋白是纯化的粘蛋白；用高碘酸/席夫试剂染色时，它们在4 - 15%梯度凝胶丙烯酰胺电泳上迁移很少。丝氨酸 + 苏氨酸 + 脯氨酸残基占总氨基酸的35%；碳水化合物组成包括半乳糖、岩藻糖、N - 乙酰半乳糖胺和N - 乙酰葡糖胺。这些粘液糖蛋白带有660表位。二硫键还原后，剩余的高分子量亚基被Ultrogel A4柱保留；氨基酸和糖类组成通常与未还原级分相似。对660表位糖蛋白载体进行胰蛋白酶消化并未改变其色谱和电泳图谱，也未改变其化学组成。这些处理后660表位仍然存在。然而，对亚基进行胰蛋白酶消化产生了被Ultrogel A4柱保留的较小成分。这些片段的糖类组成未改变，但丝氨酸 + 苏氨酸 + 脯氨酸残基的比例上升至总氨基酸的46%。这些消化后的亚基几乎失去了与单克隆抗体660的所有反应性。我们的结果与Carlstedt、Lindgren和Sheehan [(1983) Biochem. J. 213, 427 - 435]的大分子模型非常吻合：粘蛋白糖蛋白是通过二硫键端对端组装成非常大的线性大分子的亚基的同聚物。二硫键还原后，蛋白水解攻击位点被暴露，胰蛋白酶消化产生带有典型寡糖单元且丝氨酸、苏氨酸和脯氨酸含量增加的糖肽，这些是肽核心这个高糖基化区域的特征氨基酸。这些消化后的亚基几乎失去了所有660表位反应性。因此我们表明，660表位作为粘蛋白分子的一个决定簇，可能与糖蛋白的肽核心相关。
