Establishment of Detection Systems and Expression Systems for Organ-Specific Mucins

本课题的目的是建立检测各种人上皮粘蛋白的系统，并建立获得表达粘蛋白核心多肽cDNA的稳定转染细胞的系统。具体而言，制备特异性粘蛋白单克隆抗体，制备含粘蛋白核心多肽cDNA的表达载体。阐明了粘蛋白核心多肽的转录调控。以下是项目期间获得的研究结果：(1)建立了一种新的单克隆抗体MY.1E12，该抗体针对唾液化MUC1粘蛋白。(2)制备了嵌合体粘蛋白MUC1/MMGL和MUC2/MMGL载体。(3)利用人肾癌细胞制备了稳定的转染细胞系和克隆。研究了这些细胞的生物学行为。(4)转染K562人红白血病细胞表达的MUC1粘蛋白的糖基化与内源性白细胞粘蛋白的糖基化进行比较。(5)分离出一株肠杆菌表达的凝集素，发现它能结合MUC2粘蛋白，但不能结合MUC1粘蛋白。从这种细菌中分离出来的一种新物质。(7) MUC1粘蛋白基因的诱导转录顺式作用元件已被发现。从乳腺癌细胞的核提取物中分离出该元件的结合蛋白。(8)克隆并表达了与人巨噬细胞凝集素相对应的cDNA。这种凝集素的碳水化合物结合特异性已被证明与Tn和唾液-Tn抗原结合。

The aims of this proposal is to establish systems to detect various human epithelial mucins and to establish systems to obtain stable transfectant cells expressing mucin core polypeptide cDNA.Specifically, preparation of monoclonal antibodies specific for unique mucins and preparation of expression vectors containing mucin core polypeptide cDNA were conducted. Elucidation of the transcriptional regulation of mucin core polypeptides was also performed.The following list consists of the findings obtained during the project term : (1) A novel monoclonal antibody MY.1E12 specific for sialylated MUC1 mucin was established. (2) Vectors containing chimera mucins (MUC1/MMGL and MUC2/MMGL) have been prepared. (3) A stable transfectant cell line and clones have been prepared using human renal carcinoma cells. The biological behavior of these cells were investigated. (4) Glycosylation of MUC1 mucins expressed by transfection of K562 human erythroleukemia cells was compared to the glycosylation of endogenous leukocyte mucins. (5) A lectin expressed by a strain of enterobacterium was isolated and shown to bind MUC2 mucin but not MUC1 mucin. (6) A novel lecting that was isolated from this bacterium. (7) An inducible transcriptional cis-acting element for MUC1 mucin gene has been identified. Binding proteins to this element were isolated from nuclear extracts of mammary carcinoma cells. (8) A cDNA corresponding to human macrophage lectin has been cloned and expressed. The carbohydrate binding specificity of this lectin has been demonstrated to bind to Tn and sialyl-Tn antigens.