Osinaga E, Pancino G, Porchet N, Berois N, De Cremoux P, Mistro D, Aubert J P, Calvo F, Roseto A
Division D'Immuno-Cytologie Appliquée, CNRS URA 1442, Université de Technologie de Compiègne, France.
Breast Cancer Res Treat. 1994;32(2):139-52. doi: 10.1007/BF00665765.
The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.
Tn 决定簇（N-乙酰半乳糖胺α-O-丝氨酸/苏氨酸）在约 90%的人类癌组织中表达，但在大多数正常人体组织中是隐蔽的。用人类乳腺癌细胞免疫后产生的鼠单克隆抗体（MAb）83D4 与一个 Tn 相关表位发生反应。在本研究中，我们对人乳腺癌细胞系 MCF-7 中由 83D4 识别的糖蛋白抗原进行了表征。我们进一步表明，83D4 抗原决定簇在人乳脂肪球膜（HMFGM）中被掩盖，并且在去唾液酸化后经轻度高碘酸盐处理可暴露出来。蛋白质印迹分析将来自 MCF-7 的 83D4 抗原解析为分别为 120 - 190 kD 和 > 500 kD 的两个主要成分。非平衡 pH 梯度电泳/SDS - PAGE 显示反应性糖蛋白的酸性性质（pI 4.43 - 4.70）。通过 CsCl 梯度超速离心在广泛的密度范围（1.30 - 1.46 g/ml）分层解析的 83D4 抗原活性，包括粘蛋白样糖蛋白的典型密度但也有较低密度。该抗原的氨基酸组成相对富含丝氨酸但苏氨酸和脯氨酸含量低，证实了其与其他粘蛋白样癌相关糖蛋白的差异。二羧酸氨基酸丰富，部分解释了分子的酸性性质。对 MCF-7 细胞亚细胞组分的 ELISA 和蛋白质印迹分析表明，83D4 抗原主要存在于质膜中（85%），从中可解析为两条宽条带（慢迁移组分和快迁移组分）。这些结果提供了关于一组与典型粘蛋白相关但不同的乳腺癌相关糖蛋白的信息，并提供了肿瘤细胞中 O-糖基化改变的数据。
