The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.

高尔基体（Golgi apparatus）对于分泌性货物的转运发挥关键作用。糖基化是一类主要的翻译后修饰事件，识别蛋白质上的O-聚糖（O-glycans）是糖蛋白转运的必要条件。本研究中，对O-糖基化的特异性抑制（即高尔基体应激）可诱导NIH3T3细胞中内质网（ER）驻留型热休克蛋白（HSP）47的表达，尽管仅高尔基体应激本身并不会引发细胞死亡。当通过小干扰RNA（siRNA）下调HSP47的表达后，抑制O-糖基化便会引发细胞死亡。诱导高尔基体应激3天后，高尔基体发生解离，大量液泡在高尔基体附近出现并向细胞质内延伸，细胞核发生裂解，同时出现细胞死亡检测阳性的细胞。诱导高尔基体应激6小时后，HSP47敲低细胞中高尔基体驻留型半胱天冬酶（caspase）-2的剪切激活水平显著升高。此外，在诱导高尔基体应激24小时后的HSP47敲低细胞中，可观察到线粒体半胱天冬酶-9的激活、内质网驻留型未折叠蛋白反应（UPR）相关分子的活化，以及细胞色素c从线粒体向细胞质的外流。上述研究结果表明：① 内质网驻留型分子伴侣HSP47可保护细胞免受高尔基体应激的损伤；② 因HSP47表达受抑制而引发的高尔基体应激诱导型细胞死亡，源于高尔基体中半胱天冬酶-2的激活，并进一步波及内质网与线粒体。