Ratcliffe A, Fryer P R, Hardingham T E
J Cell Biol. 1985 Dec;101(6):2355-65. doi: 10.1083/jcb.101.6.2355.
The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.
利用蛋白A-金电子显微镜免疫定位技术，在分离的软骨细胞中研究了软骨蛋白聚糖生物合成的细胞内途径。蛋白聚糖含有一个蛋白质核心，在翻译后加工过程中，硫酸软骨素、硫酸角质素链和寡糖会添加到该核心上。在本研究中，使用特异性抗体分别确定蛋白质核心和硫酸软骨素成分的分布。在正常软骨细胞中，蛋白聚糖蛋白质核心仅易于定位在与蓖麻毒素共标记的光滑膜泡中，这表明它们是富含半乳糖的中间/反式高尔基体池，而粗面内质网中的标记水平较低。硫酸软骨素也定位在对照软骨细胞的中间/反式高尔基体池中，但在其他细胞区室中未检测到。在用莫能菌素（高达1.0微摩尔）处理的细胞中，莫能菌素强烈抑制蛋白聚糖分泌（Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247 - 255），蛋白聚糖蛋白质核心在蓖麻毒素阳性小泡、蓖麻毒素阴性小泡（源自顺式高尔基体堆叠）和扩张的粗面内质网中的细胞内定位都大大增加。莫能菌素处理后硫酸软骨素的丰度也增加，但仍仅定位在蓖麻毒素阳性小泡中。结果表明，蛋白聚糖上硫酸软骨素的合成仅发生在中间/反式高尔基体池中，是蛋白聚糖生物合成中的晚期事件。这也表明，蛋白聚糖上糖胺聚糖的合成发生在一个与其他分泌性糖蛋白上O-连接和N-连接寡糖生物合成事件相同的区室中。
