Vertel B M, Barkman L L
Coll Relat Res. 1984 Jan;4(1):1-20. doi: 10.1016/s0174-173x(84)80025-4.
Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L , which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D , and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.
几种识别硫酸软骨素蛋白聚糖不同抗原决定簇的单克隆抗体被用于研究鸡软骨细胞培养物中硫酸软骨素蛋白聚糖的生物合成。通过双重免疫荧光反应确定了合成和加工的细胞内位点以及在基质中的细胞外沉积。一种大鼠单克隆抗体S103L，它识别鸡软骨硫酸软骨素蛋白聚糖单体核心蛋白的一个抗原决定簇，用于鉴定细胞外硫酸软骨素蛋白聚糖和含有硫酸软骨素蛋白聚糖前体的细胞内区室。用S103L进行的细胞内染色定位于核周区域，并且在一些软骨细胞中，也定位于其他一些细胞质小泡。当软骨细胞几天不喂食时，细胞内硫酸软骨素蛋白聚糖前体积累在分布于整个细胞质中的较大区室中。多克隆硫酸软骨素蛋白聚糖抗体显示出相似的染色特征。相比之下，几种单克隆抗体，包括大鼠单克隆抗体S11D和P100D，以及小鼠单克隆抗体1-B-5、3-B-3和9-A-2，不识别天然硫酸软骨素蛋白聚糖，而是仅与软骨素酶ABC消化的（和/或透明质酸酶消化的）硫酸软骨素蛋白聚糖反应。这些抗体在证明硫酸软骨素蛋白聚糖与II型胶原或纤连蛋白的细胞外共分布方面特别有用。在其他实验中，针对硫酸软骨素蛋白聚糖的单克隆抗体用于证明细胞内区室的核周亚群独特地参与了将硫酸软骨素寡糖添加到硫酸软骨素蛋白聚糖核心蛋白上。最后，使用识别硫酸角质素决定簇的小鼠单克隆抗体5-D-4，确定核周区域为硫酸角质素添加的位点。结果表明，即使对于明显含有等量细胞内硫酸软骨素蛋白聚糖的细胞，在单个软骨细胞水平上硫酸角质素合成也存在异质性。
