Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate.
Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate.
复制标题
DOI：
10.1016/s0021-9258(18)34561-7
复制
发表时间：
1982-06
期刊：
影响因子：
--
通讯作者：
E. Razin;R. Stevens;F. Akiyama;K. Schmid;K. Austen
中科院分区：
文献类型：
--
作者：
E. Razin;R. Stevens;F. Akiyama;K. Schmid;K. Austen
在刀豆蛋白A刺激的小鼠脾细胞条件培养液中，培养2周的小鼠骨髓获得了具有异染染色颗粒和表面IgE受体的分化细胞群。这些细胞被发现将大量的[35S]硫酸盐掺入到细胞内35S标记的MR蛋白多糖中，大约200,000个细胞内含有最多7个糖胺多糖侧链(MR=25,000)。经密度梯度纯化的[~3H]丝氨酸标记的蛋白多糖经软骨素酶ABC处理后，通过凝胶过滤鉴定得到的核心蛋白约为26,000个。经去β-35S标记的糖胺多聚糖的醋酸纤维素二维电泳法显示，单一类型的糖胺多聚糖在鱿鱼软骨中过硫酸软骨素E的位置迁移。软骨素酶ABC降解35S标记的糖胺聚糖产生两个摩尔数大致相等的裂解产物，它们在下行纸层析和高压纸电泳中与单磺化二糖(2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic酸)-4-O-磺酸-D-半乳糖和二硫化二糖(2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic酸)-4-O-二磺基-D-半乳糖共同迁移。用软骨-4-硫酸酯酶或软骨-6-硫酸酯酶从过硫酸化的二糖中释放出一些游离的[35S]硫酸盐，并在它们的共同作用下完全脱硫，证实了过硫化的二糖含有N-乙酰半乳糖胺-4，6-二硫酸盐。因此，这些独特的IgE受体和含有组胺的细胞的35S标记的蛋白多糖是由硫酸软骨素E而不是肝素糖胺聚糖组成的，因此这是首次在哺乳动物细胞中鉴定到这种细胞内定位的蛋白多糖。
A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.