Uhlin-Hansen L, Wik T, Kjellén L, Berg E, Forsdahl F, Kolset S O
Department of Biochemistry, University of Tromsø, Norway.
Blood. 1993 Nov 1;82(9):2880-9.
To study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopolysaccharide (LPS). When exposed to [35S]sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depolymerized (t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.
为了研究炎症巨噬细胞中的蛋白聚糖代谢，在有无细菌脂多糖（LPS）的情况下培养人巨噬细胞原代培养物。当暴露于[35S]硫酸盐时，细胞几乎将标记物全部掺入硫酸软骨素蛋白聚糖（CSPG）中，该蛋白聚糖可从培养基和细胞层中回收。用LPS刺激的细胞向培养基中分泌的[35]CSPG比对照细胞多约三倍。此外，还发现细胞黏附可促进蛋白聚糖的分泌；当诱导非黏附单核细胞黏附时，蛋白聚糖的释放增加了两倍。LPS刺激的巨噬细胞中观察到的分泌增加部分归因于生物合成增加，但主要归因于CSPG向分泌途径的分选增加。未刺激细胞中合成的CSPG只有约20%被分泌，而LPS处理细胞中的相应比例为35%。在两种细胞类型中，剩余的[35S]CSPG可能在溶酶体中被降解。降解是一个两步过程。首先，[35S]CSPG迅速裂解产生游离糖胺聚糖（GAG）链（半衰期=15至30分钟）。其次，GAG链完全解聚（半衰期=2至3小时）。静息细胞和LPS刺激的细胞都不会将CSPG分选到细胞内储存，这在许多造血细胞中很明显。LPS处理的细胞合成的[35S]CSPG分子大小比对照细胞小，GAG链的近似分子量为12 kD，而对照细胞中为16 kD。GAG链的二糖组成没有差异；LPS刺激和未刺激的细胞都表达80%至90%硫酸软骨素4和10%至20%硫酸软骨素4,6二硫酸的混合物。N端序列和Northern印迹分析表明，人巨噬细胞分泌的CSPG的核心蛋白是丝甘蛋白聚糖。
