Siddiqui B, Byrd J C, Fearney F J, Kim Y S
Gastrointestinal Research Laboratory, Veterans Administration Medical Center, San Francisco, Calif.
Tumour Biol. 1989;10(2):83-94. doi: 10.1159/000217600.
Colon cancer cells in culture synthesize and secrete mucin glycoproteins, which carry a number of cancer-associated antigens. However, the structures and mechanisms of biosynthetic processing are not well understood. Mucins synthesized and secreted by LS174T human colon cancer cells were compared to those in LS174T xenografts in athymic mice. Mucins radiolabeled with glucosamine or sulfate were purified by gel filtration and cesium chloride density gradient centrifugation. The mucins were of high molecular weight and were resistant to chondroitinase ABC, hyaluronidase and HNO2 treatment. They were, however, susceptible to pronase digestion and mild alkaline treatment. Using radiochemical precursors, the cellular mucin was shown to contain fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, and sulfate. Oligosaccharides released by beta-elimination had N-acetylgalactosaminitol as the reduced amino sugar and also unreduced galactosamine, indicating that there is N-acetyl-galactosamine O-glycosidically attached to protein core and also peripheral N-acetyl-galactosamine not directly linked to protein. DEAE-cellulose chromatography of mucins showed two major peaks with both intracellular and secreted mucins, but xenograft mucins also had more acidic components. Sulfate-labeled mucins were shifted to less acidic peaks by neuraminidase digestion, which indicates that the same mucin molecules are both sialylated and sulfated. We conclude that the intracellular mucins of cultured colon cancer cells, those secreted into the medium, and those in nude mouse xenografts are chemically similar, but differ in sialic acid and sulfate content. This experimental model system, LS174T cells maintained in culture and as nude mouse xenografts, may be useful for further biosynthetic and structural studies of colon cancer mucin.
培养的结肠癌细胞能合成并分泌粘蛋白糖蛋白，这些糖蛋白带有许多与癌症相关的抗原。然而，生物合成加工的结构和机制尚未得到充分了解。将LS174T人结肠癌细胞合成并分泌的粘蛋白与无胸腺小鼠体内LS174T异种移植瘤中的粘蛋白进行了比较。用葡糖胺或硫酸盐进行放射性标记的粘蛋白通过凝胶过滤和氯化铯密度梯度离心进行纯化。这些粘蛋白分子量高，对软骨素酶ABC、透明质酸酶和亚硝酸处理具有抗性。然而，它们易受链霉蛋白酶消化和温和碱性处理的影响。使用放射性化学前体，显示细胞粘蛋白含有岩藻糖、半乳糖、N-乙酰半乳糖胺、N-乙酰葡糖胺、N-乙酰神经氨酸和硫酸盐。通过β-消除释放的寡糖以N-乙酰半乳糖胺醇作为还原氨基糖，还含有未还原的半乳糖胺，这表明存在通过O-糖苷键连接到蛋白质核心的N-乙酰半乳糖胺以及不直接与蛋白质相连的外周N-乙酰半乳糖胺。粘蛋白的DEAE-纤维素色谱显示细胞内和分泌的粘蛋白都有两个主要峰，但异种移植瘤粘蛋白也有更多酸性成分。经神经氨酸酶消化后，硫酸盐标记的粘蛋白转移到酸性较低的峰，这表明相同的粘蛋白分子既被唾液酸化又被硫酸化。我们得出结论，培养的结肠癌细胞的细胞内粘蛋白、分泌到培养基中的粘蛋白以及裸鼠异种移植瘤中的粘蛋白在化学性质上相似，但唾液酸和硫酸盐含量不同。这个实验模型系统，即培养的LS174T细胞和作为裸鼠异种移植瘤的LS174T细胞，可能有助于进一步开展结肠癌粘蛋白的生物合成和结构研究。
