Yamori T, Ota D M, Cleary K R, Hoff S, Hager L G, Irimura T
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Cancer Res. 1989 Feb 15;49(4):887-94.
Previous studies using metabolic labeling of fresh colonic mucosa and colorectal carcinoma with [35S]sulfate followed by biochemical analysis demonstrated that the amount of a sulfated high-molecular-weight glycoprotein expressed in primary colorectal carcinoma was lower than that in normal mucosa, and that the amount further decreased in liver metastases. This suggested that this sulfated molecule represented a sulfomucin previously defined by histochemical reactivity with a cationic dye. We have extracted and partially purified this high-molecular-weight sulfated glycoprotein from normal human colonic mucosa. We immunized mice with the partially purified sulfomucin and generated hybridomas. One cloned hybridoma, designated as 91.9H, produced a monoclonal antibody strongly reactive with a component which migrated at an identical position as the metabolically [35S]sulfate-labeled high-molecular-weight glycoprotein after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reactive molecules appeared to have a polydisperse nature with a molecular weight ranging between 400,000 and 900,000. The [35S]sulfate-labeled high-molecular-weight glycoprotein was bound to Staphylococcus Protein A-agarose coated with this monoclonal antibody but did not bind to unconjugated Protein A-agarose. The immunoprecipitated substance also migrated at an apparent molecular weight range of 400,000 to 900,000. The reactivity of monoclonal antibody 91.9H with the extracts of normal mucosa, colorectal primary carcinoma, and metastasis was compared by dot blot assay on a nitrocellulose membrane. This antibody was more reactive with the extracts of mucosa adjacent to carcinoma tissues than with the carcinoma extracts. Primary tumors showed higher reactivity than metastases in most of the cases. These results strongly suggest that this antibody is specific to colonic sulfomucins or at least to mucins closely related to colonic mucins previously identified by metabolic labeling with [35S]sulfate.
以往的研究通过用[35S]硫酸盐对新鲜结肠黏膜和结直肠癌进行代谢标记，随后进行生化分析，结果表明，原发性结直肠癌中表达的一种硫酸化高分子量糖蛋白的量低于正常黏膜中的量，且在肝转移灶中该量进一步降低。这表明这种硫酸化分子代表了一种先前通过与阳离子染料的组织化学反应定义的硫黏液素。我们已从正常人结肠黏膜中提取并部分纯化了这种高分子量硫酸化糖蛋白。我们用部分纯化的硫黏液素免疫小鼠并产生了杂交瘤。一个克隆的杂交瘤，命名为91.9H，产生了一种单克隆抗体，该抗体与在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳后，与经代谢[35S]硫酸盐标记的高分子量糖蛋白迁移到相同位置的一种成分发生强烈反应。反应性分子似乎具有多分散性，分子量在400,000至900,000之间。经[35S]硫酸盐标记的高分子量糖蛋白与包被有这种单克隆抗体的葡萄球菌蛋白A -琼脂糖结合，但不与未结合的蛋白A -琼脂糖结合。免疫沉淀物质也在400,000至900,000的表观分子量范围内迁移。通过在硝酸纤维素膜上的斑点印迹分析比较了单克隆抗体91.9H与正常黏膜、结直肠癌原发性肿瘤和转移灶提取物的反应性。该抗体与癌组织相邻黏膜的提取物的反应性高于与癌提取物的反应性。在大多数情况下，原发性肿瘤的反应性高于转移灶。这些结果强烈表明，该抗体对结肠硫黏液素具有特异性，或者至少对与先前通过[35S]硫酸盐代谢标记鉴定的结肠黏蛋白密切相关的黏蛋白具有特异性。
