Vertel B M, Barkman L L, Morrell J J
J Cell Biochem. 1985;27(3):215-29. doi: 10.1002/jcb.240270304.
The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.
利用同步双重免疫荧光和凝集素定位反应，研究了参与II型前胶原和硫酸软骨素蛋白聚糖（CSPG）单体合成与加工的软骨细胞内区室。II型前胶原分布于整个细胞质的囊泡中，而CSPG单体的细胞内前体则积聚在核周细胞质中。在本研究中，还观察到了胞质囊泡，这些囊泡与针对CSPG单体的抗体发生强烈反应，但不与II型胶原抗体反应。一种识别硫酸角质素决定簇的单克隆抗体5-D-4用于鉴定高尔基体（硫酸角质素链延长的部位）。用5-D-4染色仅限于核周细胞质。核周细胞质外与CSPG单体抗体发生强烈反应的囊泡不与5-D-4反应。荧光凝集素用于进一步表征亚细胞区室。与富含甘露糖的寡糖反应的伴刀豆球蛋白A不染色核周区域，但它确实染色了细胞质其余部分的囊泡。由于甘露糖寡糖是共翻译添加的，因此整个细胞质中染色的囊泡可能对应于粗面内质网。识别N-乙酰-D-葡萄糖胺和唾液酸（在高尔基体中添加的碳水化合物）的麦胚凝集素仅染色核周细胞质。根据几个标准（用单克隆抗体5-D-4和麦胚凝集素染色），核周细胞质似乎对应于高尔基体。与抗CSPG单体反应而不与抗II型胶原反应的胞质囊泡含有尚未经高尔基体介导的寡糖添加修饰的CSPG单体前体（因为它们未被麦胚凝集素或抗硫酸角质素抗体染色）；这些囊泡可能在CSPG的加工过程中具有独特的功能。
