Mueller W H, Kleefeld D, Khattab B, Meissner J D, Scheibe R J
Zentrum Biochemie, Medizinische Hochschule Hannover, Hannover, Germany.
J Cell Physiol. 2000 Jan;182(1):50-61. doi: 10.1002/(SICI)1097-4652(200001)182:1<50::AID-JCP6>3.0.CO;2-6.
Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.
碱性磷酸酶（ALP）是一种糖酶，在致癌过程中高度表达，并在各种细胞中由视黄酸（RA）诱导产生。我们研究了RA对组织非特异性肝/骨/肾型碱性磷酸酶（L/B/K ALP）的N-连接糖基化、碱性磷酸酶转录本以及两种神经细胞系P19和NG108CC15以及新生大鼠脑原代培养物中总蛋白糖基化的影响。在细胞提取物中测定了碱性磷酸酶活性，发现其可被RA诱导。使用不同浓度的衣霉素抑制蛋白质N-糖基化。用低浓度（0.1和0.125微克/毫升）的衣霉素处理细胞48小时后，未诱导和RA诱导的碱性磷酸酶活性均下降，而与蛋白酶抑制剂一起孵育可恢复活性，这表明L/B/K ALP带有对维持酶活性很重要的N-连接寡糖链。有趣的是，与未处理的对照相比，RA处理培养物中的碱性磷酸酶活性受衣霉素的抑制较小，这表明RA可能对碱性磷酸酶的N-糖基化有影响。为了进一步研究RA对碱性磷酸酶糖基化的影响，在有或没有RA的情况下测定了[(14)C]甘露糖和[(35)S]甲硫氨酸掺入碱性磷酸酶蛋白的情况。甘露糖基化与生物合成的比率表明，用RA孵育细胞会增加[(14)C]甘露糖掺入碱性磷酸酶分子的量。此外，在RA处理的培养物中，相对于蛋白量，碱性磷酸酶分子释放的游离[(14)C]甘露糖通过N-聚糖酶增加。另外，发现暴露于RA后细胞中总蛋白的甘露糖基化被诱导。对生物合成的碱性磷酸酶单体的分析表明，RA增加了多肽的糖基化。此外，衣霉素降低了碱性磷酸酶mRNA的稳定性，而与RA共同处理可降低这种作用。因此，L/B/K ALP的N-糖基化程度以及该酶的mRNA和蛋白水平受RA影响。P19细胞系为进一步研究RA在神经元分化过程中对糖基化作用的分子机制提供了一个有用的模型系统。
