Crestani B, Dehoux M, Seta N, Cuer M, Aubier M
INSERM U 226, Faculté Xavier Bichat, Paris, France.
Am J Respir Cell Mol Biol. 1993 Feb;8(2):145-52. doi: 10.1165/ajrcmb/8.2.145.
Cell surface carbohydrates have been shown to be altered during cellular differentiation. Alveolar type II (ATII) cells in culture gradually lose their differentiated phenotype. Therefore, the aim of this study was: (1) to characterize changes in terminal carbohydrates of cell surface glycoproteins of rat ATII cells cultured for 1 to 5 days on plastic, and (2) to assess the concomitant changes in sialidase and sialyltransferase activity of ATII cell homogenates. Cells were surface-labeled with potassium-[3H]-borohydride after oxidation by sodium periodate at millimolar concentrations, galactose oxidase or neuraminidase plus galactose oxidase, allowing for the specific labeling of terminal sialic acids, terminal galactose/N-acetylgalactosamine (Gal/GalNAc), or terminal an penultimate Gal/GalNAc residues, respectively. Glycoproteins were separated by SDS-PAGE. On day 1, cells were heavily coated with sialic acids, since no labeling could be introduced with galactose oxidase alone. From day 1 to day 5, we observed a selective and progressive desialylation of two glycoproteins (200 and 165 kD). At the same time, the ATII cells' sialidase activity (pH 4.2) exhibited an 8-fold increase (60.3 +/- 4.0 pmol/min/mg protein on day 1 versus 406.9 +/- 3.7 pmol/min/mg protein on day 5), whereas the sialyltransferase activity increased 2-fold (212 +/- 8 fmol/min/mg protein on day 1 versus 395 +/- 82 fmol/min/mg protein on day 5) and the supernatant sialidase activity was unchanged (2.8 +/- 0.7 pmol/min/ml on day 5). Thus, the phenotypic changes of ATII cells in primary culture are accompanied by a partial cell surface desialylation and an increase in intracellular sialidase activity.
细胞表面碳水化合物在细胞分化过程中会发生改变。培养的II型肺泡（ATII）细胞会逐渐失去其分化表型。因此，本研究的目的是：（1）表征在塑料上培养1至5天的大鼠ATII细胞表面糖蛋白末端碳水化合物的变化，以及（2）评估ATII细胞匀浆中唾液酸酶和唾液酸转移酶活性的相应变化。细胞在经毫摩尔浓度的高碘酸钠氧化后，用[3H] -硼氢化钾进行表面标记，或用半乳糖氧化酶、或神经氨酸酶加半乳糖氧化酶处理，分别实现对末端唾液酸、末端半乳糖/N-乙酰半乳糖胺（Gal/GalNAc）或末端及倒数第二个Gal/GalNAc残基的特异性标记。糖蛋白通过SDS-PAGE分离。在第1天，细胞被大量唾液酸覆盖，因为单独用半乳糖氧化酶无法引入标记。从第1天到第5天，我们观察到两种糖蛋白（200和165 kD）出现选择性且渐进性的去唾液酸化。与此同时，ATII细胞的唾液酸酶活性（pH 4.2）增加了8倍（第1天为60.3±4.0 pmol/分钟/毫克蛋白，第5天为406.9±3.7 pmol/分钟/毫克蛋白），而唾液酸转移酶活性增加了2倍（第1天为212±8 fmol/分钟/毫克蛋白，第5天为395±82 fmol/分钟/毫克蛋白），上清液唾液酸酶活性未变（第5天为2.8±0.7 pmol/分钟/毫升）。因此，原代培养中ATII细胞的表型变化伴随着部分细胞表面去唾液酸化以及细胞内唾液酸酶活性的增加。
