Forsberg L S, Lazarus S C, Seno N, DeVinney R, Caughey G H, Gold W M
Cardiovascular Research Institute, University of California, San Francisco 94143.
Biochim Biophys Acta. 1988 Dec 15;967(3):416-28. doi: 10.1016/0304-4165(88)90105-5.
The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.
分离了三种犬肥大细胞瘤细胞系合成的细胞相关蛋白聚糖，并比较了它们的结构特征。这些细胞系在无胸腺小鼠中作为皮下肿瘤传代培养超过25代。在原代细胞培养中，所有三种细胞系都将[35S]硫酸盐掺入到大小和糖胺聚糖含量均异质的高分子量蛋白聚糖中。两个细胞系BR和G以不同比例合成了肝素蛋白聚糖（HPG）和硫酸软骨素蛋白聚糖（ChSPG）。第三个细胞系C2主要合成ChSPG，几乎检测不到肝素或没有肝素。在解离条件（4M胍，Triton X-100）下，将来自BR细胞系的35S标记的HPG和ChSPG在Sepharose CL-4B上进行凝胶过滤，得到一个主要的多分散峰（Kav = 0.22），占35S活性的70％。在Sepharose CL-4B上的聚集条件（0.1M醋酸钠）下，BR蛋白聚糖在排阻体积中洗脱。在非解离条件下，来自G和C2细胞系的蛋白聚糖也在空体积中洗脱，但是C2细胞系产生了另外一些流体力学尺寸较小的级分（Kav = 0.81），表明存在细胞内蛋白聚糖裂解产物或未完全加工的蛋白聚糖。通过在Sepharose CL-4B上的解离色谱法评估，在降解大鼠软骨肉瘤蛋白聚糖的条件下，BR细胞系的蛋白聚糖对蛋白酶裂解具有抗性。对于所有细胞系，链霉蛋白酶/碱性硼氢化物释放的糖胺聚糖在凝胶过滤时分子量范围为20,000至50,000。对于BR细胞系，75％的35S标记的糖胺聚糖被亚硝酸降解为寡糖，其余25％被软骨素酶ABC降解。G细胞系的相应百分比为89％和11％，C2细胞系为2％和98％。BR和C2细胞系的软骨素酶消化产物的纸色谱显示，除了少量单硫酸化二糖外，产物对应于不饱和标准品δDi-diSB和δDi-diSE，分别衍生自二糖艾杜糖醛酸-2-硫酸酯---- N-乙酰半乳糖胺-4-硫酸酯和葡萄糖醛酸---- N-乙酰半乳糖胺-4,6-二硫酸酯。C2和BR细胞系的聚糖含有少量三硫酸化二糖，与软骨素-6-硫酸酯酶孵育后降解为δDi-diSB。结果表明，克隆来源的犬肥大细胞中同时存在肝素和多硫酸化硫酸软骨素。
