Zanghi J A, Mendoza T P, Knop R H, Miller W M
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208-3120, USA.
J Cell Physiol. 1998 Nov;177(2):248-63. doi: 10.1002/(SICI)1097-4652(199811)177:2<248::AID-JCP7>3.0.CO;2-N.
Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large-scale production systems can parallel those that develop within fast-growing solid tumors such as small cell lung cancer (SCLC). Ammonia's specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane-bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH4Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose-dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI-N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4-day culture with 10 mM NH4Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N41 7 cells is a global rather than glycosylation-specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression.
氨是生物技术领域的一个主要问题，因为它常常限制动物细胞生产重组蛋白。在大规模生产系统中，诸如氨积累等情况可能与快速生长的实体瘤（如小细胞肺癌（SCLC））内部出现的情况相似。氨对分泌型糖蛋白唾液酸化的特异性抑制作用已有充分记录，但尚不清楚氨如何影响膜结合蛋白，也不清楚它在癌症中重要的糖基化决定因素上可能发挥什么作用。因此，我们研究了氯化铵对神经细胞黏附分子（NCAM）中多唾液酸（PolySia）的影响。通过使用流式细胞术结合两种NCAM抗体，一种针对肽骨架，另一种识别PolySia链，我们发现氨会导致中国仓鼠卵巢（CHO）细胞和SCLC NCI - N417细胞中NCAM多唾液酸化的快速、剂量依赖性和可逆抑制。PolySia的减少伴随着NCAM的小幅增加，这表明这些变化是寡糖特有的。氨对CHO细胞的抑制作用更强，在含有10 mM氯化铵的培养基中培养4天后，PolySia细胞表面含量降至对照的10%，而N417细胞的PolySia仅减少35%。氨使CHO细胞从葡萄糖产生的产量降低了60%，而N417细胞几乎不受影响，这表明N417细胞对氨的抗性增加是一种整体现象，而非糖基化特异性现象。所呈现的数据表明，肿瘤微环境可能是调节PolySia表达的一个重要因素。
