Razin E, Stevens R L, Akiyama F, Schmid K, Austen K F
J Biol Chem. 1982 Jun 25;257(12):7229-36.
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.
在伴刀豆球蛋白A刺激的脾细胞来源的条件培养基存在下，对小鼠骨髓进行2周培养，获得了一群具有异染性染色颗粒和表面IgE受体的分化细胞。发现这些细胞将大量的[35S]硫酸盐掺入到一种细胞内35S标记的蛋白聚糖中，该蛋白聚糖的Mr约为200,000，最多含有7条糖胺聚糖侧链（Mr = 25,000）。用软骨素酶ABC处理密度梯度纯化的[3H]丝氨酸标记的蛋白聚糖后，通过凝胶过滤评估所得核心的Mr约为26,000。对β消除的35S标记的糖胺聚糖进行二维醋酸纤维素电泳，显示出一种单一类型的糖胺聚糖，其迁移位置与鱿鱼软骨中过硫酸化硫酸软骨素E的位置相同。35S标记的糖胺聚糖经软骨素酶ABC降解产生两种摩尔量大致相等的裂解产物，它们在下行纸色谱和高压纸电泳中与单硫酸化二糖2-乙酰氨基-2-脱氧-3-O-（β-D-葡萄糖-4-烯吡喃糖醛酸）-4-O-磺基-D-半乳糖和二硫酸化二糖2-乙酰氨基-2-脱氧-3-O-（β-D-葡萄糖-4-烯吡喃糖醛酸）-4,6-二-O-磺基-D-半乳糖共迁移。用过硫酸化二糖与软骨素-4-硫酸酯酶或软骨素-6-硫酸酯酶反应释放出一些游离的[35S]硫酸盐，以及它们联合作用导致的完全脱硫，证实过硫酸化二糖含有N-乙酰半乳糖胺-4,6-二硫酸盐。因此，这些独特的带有IgE受体和含组胺的细胞的35S]标记蛋白聚糖由硫酸软骨素E而非肝素糖胺聚糖组成，这是首次在哺乳动物细胞中鉴定出这种细胞内定位的蛋白聚糖。
