Hilkens J, Buijs F, Ligtenberg M
Division of Tumor Biology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Amsterdam.
Cancer Res. 1989 Feb 15;49(4):786-93.
The complexity of epithelial sialomucins was investigated by immunoprecipitation and membrane immunofluorescence, using monoclonal antibodies (MAbs) against MAM-6 and other sialomucins. MAbs against MAM-6 immunoprecipitated from a variety of sources either one or two sialylated glycoproteins with apparent molecular weights of over 400,000 under reducing as well as nonreducing conditions. The electrophoretic mobility of each MAM-6 glycoprotein as isolated from serum, milk, and cell lines of different individuals showed considerable variation. The differences in molecular weight of the MAM-6 glycoproteins were also reflected at the level of MAM-6 precursors which are less heavily glycosylated. Therefore, large differences in apparent molecular weight (150,000 and over) are most likely due to a variable protein backbone. We used this molecular polymorphism to prove that 11 MAbs against different sialomucins, obtained from various investigators, precipitated sialomucins generated from common precursor molecules. The pattern of reactivity of the MAbs with carcinoma cell lines was complex. All but the two MAbs, directed against putative carbohydrate epitopes, immunoprecipitated the precursor molecule from each cell line. However, some of them were unable to immunoprecipitate the mature form of MAM-6 from these cell lines. These results indicate that those epitopes are masked, probably due to cell line- or possibly cell type-dependent variations in glycosylation of the epithelial sialomucin. Even within a single cell line mature molecules with different epitopes were observed. The differential reactivity of the MAbs was confirmed by membrane immunofluorescence. These results show that MAM-6 belongs to a family of epithelial sialomucins with a polymorphic protein backbone and extensive variation in glycosylation.
利用针对MAM-6和其他涎粘蛋白的单克隆抗体（MAb），通过免疫沉淀和膜免疫荧光法研究了上皮涎粘蛋白的复杂性。针对MAM-6的单克隆抗体在还原和非还原条件下，从多种来源免疫沉淀出一种或两种表观分子量超过400,000的唾液酸化糖蛋白。从不同个体的血清、乳汁和细胞系中分离出的每种MAM-6糖蛋白的电泳迁移率显示出相当大的差异。MAM-6糖蛋白分子量的差异在糖基化程度较低的MAM-6前体水平也有体现。因此，表观分子量（150,000及以上）的巨大差异很可能是由于可变的蛋白质主链造成的。我们利用这种分子多态性证明，从不同研究者处获得的11种针对不同涎粘蛋白的单克隆抗体沉淀出了由共同前体分子产生的涎粘蛋白。单克隆抗体与癌细胞系的反应模式很复杂。除了两种针对假定碳水化合物表位的单克隆抗体外，所有抗体都从每个细胞系中免疫沉淀出了前体分子。然而，其中一些抗体无法从这些细胞系中免疫沉淀出成熟形式的MAM-6。这些结果表明，这些表位被掩盖了，可能是由于上皮涎粘蛋白糖基化的细胞系或可能的细胞类型依赖性变化所致。即使在单个细胞系中也观察到了具有不同表位的成熟分子。膜免疫荧光证实了单克隆抗体的差异反应性。这些结果表明，MAM-6属于一个上皮涎粘蛋白家族，其蛋白质主链具有多态性，糖基化存在广泛差异。
