Santhanam U, Ghrayeb J, Sehgal P B, May L T
Rockefeller University, New York, New York 10021.
Arch Biochem Biophys. 1989 Oct;274(1):161-70. doi: 10.1016/0003-9861(89)90427-x.
We have previously reported that interleukin (IL)-6 secreted by human fibroblasts induced with either IL-1 or tumor necrosis factor (TNF) consists of at least six differentially modified phosphoglycoproteins of molecular mass 23-30 kDa: a triplet in the mass range from 23 to 25 kDa and another triplet in the range from 28 to 30 kDa. We now report that a combination of metabolic labeling, glycosidase digestion, and lectin chromatography experiments demonstrates that the 23- to 25-kDa species are O-glycosylated and that the 28- to 30-kDa species are both O- and N-glycosylated. Pulse-chase experiments reveal that newly synthesized IL-6 polypeptides rapidly enter two separate protein modification pathways: one leads to O-glycosylation and the other to both N- and O-glycosylation; polypeptides in both pathways are further modified (phosphorylation) prior to secretion. Although both pathways appear to be equally utilized in IL-1- or TNF-induced fibroblasts, the relative proportion of polypeptides proceeding through one or the other pathway can be experimentally modified. In the presence of tunicamycin, IL-6 is secreted exclusively in the O-glycosylated form, whereas in the presence of cycloheximide the pathway leading to both N- and O-glycosylation is dominant. The inclusion of monensin (1 microM) does not inhibit IL-6 secretion from fibroblasts even though it inhibits glycosylation. Combined immunoprecipitation, immunoblotting, and immunoaffinity chromatography experiments reveal additional IL-6 species with mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions corresponding to molecular masses 17-19 kDa and 45 kDa, suggesting that this cytokine undergoes further alterations. These observations highlight an aspect of IL-6 biosynthesis that appears to represent an excellent model system for studying the mechanisms regulating post-translational protein modifications in human cells and also suggest a basis for reconciling conflicting descriptions of IL-6 structure.
我们之前报道过，用白细胞介素-1（IL-1）或肿瘤坏死因子（TNF）诱导的人成纤维细胞分泌的白细胞介素-6（IL-6）至少由六种分子量为23 - 30 kDa的差异修饰磷酸糖蛋白组成：一个分子量范围在23至25 kDa的三重态和另一个在28至30 kDa范围内的三重态。我们现在报道，代谢标记、糖苷酶消化和凝集素色谱实验的组合表明，23至25 kDa的物种是O-糖基化的，而28至30 kDa的物种是O-和N-糖基化的。脉冲追踪实验表明，新合成的IL-6多肽迅速进入两条独立的蛋白质修饰途径：一条导致O-糖基化，另一条导致N-和O-糖基化；两条途径中的多肽在分泌前都会进一步修饰（磷酸化）。虽然两条途径在IL-1或TNF诱导的成纤维细胞中似乎都被同等利用，但通过一条或另一条途径的多肽的相对比例可以通过实验进行改变。在衣霉素存在的情况下，IL-6仅以O-糖基化形式分泌，而在环己酰亚胺存在的情况下，导致N-和O-糖基化的途径占主导。即使莫能菌素（1 microM）抑制糖基化，但它并不抑制成纤维细胞分泌IL-6。联合免疫沉淀、免疫印迹和免疫亲和色谱实验揭示了在还原条件下十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中迁移率对应于分子量17 - 19 kDa和45 kDa的其他IL-6物种，表明这种细胞因子会发生进一步的改变。这些观察结果突出了IL-6生物合成的一个方面，这似乎代表了一个研究人类细胞中翻译后蛋白质修饰调控机制的优秀模型系统，也为协调对IL-6结构的相互矛盾的描述提供了一个基础。
