el Battari A, Forget P, Fouchier F, Pic P
CNRS URA 202, Université d'Aix-Marseille, France.
Biochem J. 1991 Sep 1;278 ( Pt 2)(Pt 2):527-33. doi: 10.1042/bj2780527.
We used inhibitors of four steps of the glycosylation pathway to examine the contribution of carbohydrate moieties to the ligand-binding activity, cell-surface expression and apparent molecular mass of the human vasoactive intestinal peptide (VIP) receptor. Human melanoma IGR 39 cells, incubated for 60 h with the inhibitors tunicamycin, castanospermine, swainsonine or deoxymannojirimycin, under conditions where cell viability and protein synthesis were not affected, expressed VIP receptor species with different VIP-binding properties. The most pronounced effects on VIP binding were obtained with tunicamycin and deoxymannojirimycin, which respectively caused 80% and 67% inhibition. Treatment with either swainsonine or castanospermine resulted in only a 25-32% decrease in VIP specific binding. Based on Scatchard analyses of data from competition experiments, the decrease in VIP-binding activity in either swainsonine- or deoxymannojirimycin-treated cells was due to a decrease in ligand affinity; the cell-surface number of VIP-binding sites remained unchanged. In contrast, tunicamycin and castanospermine caused decreases in the cell-surface number of functional VIP receptors without affecting affinity. Besides, the drug-treated cells produced VIP-binding proteins with different molecular masses and endoglycosidase H (Endo H) sensitivities. When compared with their counterpart synthesized in control cells, VIP-binding proteins produced by deoxymannojirimycin- or swainsonine-treated cells were smaller in size and exhibited the expected sensitivity to Endo H. No modification in the apparent molecular mass was observed in the presence of either castanospermine or tunicamycin. In addition, after Endo F digestion, all of the deglycosylated proteins migrated with the same electrophoretic mobility. Finally, processing in the presence of castanospermine led to an Endo H-resistant receptor species which showed an unexpected neuraminidase-sensitivity, indicating that, as in control cells, these receptors carry V-linked oligosaccharides with terminal sialic acid residues.
我们使用糖基化途径四个步骤的抑制剂，来研究碳水化合物部分对人血管活性肠肽（VIP）受体的配体结合活性、细胞表面表达及表观分子量的影响。在细胞活力和蛋白质合成不受影响的条件下，用抑制剂衣霉素、栗精胺、苦马豆素或脱氧甘露基野黾草素处理人黑色素瘤IGR 39细胞60小时，这些细胞表达出具有不同VIP结合特性的VIP受体种类。衣霉素和脱氧甘露基野黾草素对VIP结合的影响最为显著，分别导致80%和67%的抑制。用苦马豆素或栗精胺处理仅使VIP特异性结合降低25 - 32%。基于竞争实验数据的Scatchard分析，苦马豆素或脱氧甘露基野黾草素处理的细胞中VIP结合活性的降低是由于配体亲和力下降；VIP结合位点的细胞表面数量保持不变。相反，衣霉素和栗精胺导致功能性VIP受体的细胞表面数量减少，而不影响亲和力。此外，药物处理的细胞产生了具有不同分子量和内切糖苷酶H（Endo H）敏感性的VIP结合蛋白。与在对照细胞中合成的对应物相比，脱氧甘露基野黾草素或苦马豆素处理的细胞产生的VIP结合蛋白尺寸更小，并表现出对Endo H的预期敏感性。在栗精胺或衣霉素存在的情况下，未观察到表观分子量的改变。此外，经Endo F消化后，所有去糖基化蛋白以相同的电泳迁移率迁移。最后，在栗精胺存在下进行加工导致产生一种对Endo H有抗性的受体种类，该种类显示出意外的神经氨酸酶敏感性，表明与对照细胞一样，这些受体携带带有末端唾液酸残基的V - 连接寡糖。
