Klein D J, Brown D M, Kim Y, Oegema T R
University of Minnesota, Department of Pediatrics, Minneapolis 55455.
J Biol Chem. 1990 Jun 5;265(16):9533-43.
Human fetal kidney mesangial cells were cultured for 24 h in the presence of 3H-amino acids and [35S] sulfate and chased for 24 h in nonradioactive medium. Incubation medium and cell layer proteoglycans were purified twice by high performance liquid chromatography-DEAE chromatography followed by gel filtration chromatography. The major medium 35S-macromolecules were chondroitin/dermatan-35SO4 proteoglycans. A small, Sepharose CL-6B Kav 0.14 dermatan-35SO4 proteoglycan was detected in the labeling medium and was released into both the early (time 0-0.5 h) and late (6-24 h) chase media. It contained 38 kDa 4-sulfated 35S-GAGs with a high content of iduronic acid and a 45-kDa protein core. A protein core of similar molecular weight was detected in the culture medium by Western analysis using antibodies to biglycan or proteoglycan-I (Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571-4576). This 35S-proteoglycan was not detected in the cell layer. However, a small dermatan-35SO4 with little or no protein core was present in the intracellular compartment. A large, Sepharose CL-6B excluded chondroitin-35SO4 proteoglycan was released into the culture medium and was detected between 6 and 24 h in chase medium. It eluted near the void volume of both associative and dissociative Sepharose CL-4B columns. It contained 30-kDa 4- and 6-sulfated 35S-GAGs and a 253-kDa protein core. A chondroitin-35SO4 proteoglycan with similar sized 35S-GAGs was detected in both the detergent-soluble and insoluble cell layer compartments. A Sepharose CL-6B Kav 0.11 heparin-35SO4 proteoglycan with a 220-kDa protein core and 38-kDa 35S-GAGs was rapidly released from the cell layer. This proteoglycan was larger than that previously described in isolated rat glomeruli or glomerular basement membranes, but had a core protein similar in size to one previously detected in these tissues. A larger heparan-35SO4 proteoglycan with larger 35S-GAGs was present in the detergent-insoluble cell layer compartment. The proteoglycans released by glomerular mesangial cells in culture resembled those synthesized by aortic smooth muscle cells in culture or extracted from aorta, supporting the notion that these cells are of vascular origin.
人胎儿肾系膜细胞在含有³H - 氨基酸和[³⁵S]硫酸盐的条件下培养24小时，然后在无放射性培养基中进行24小时的追踪培养。孵育培养基和细胞层蛋白聚糖通过高效液相色谱 - DEAE色谱，随后进行凝胶过滤色谱纯化两次。主要的培养基³⁵S - 大分子是硫酸软骨素/硫酸皮肤素 - ³⁵SO₄蛋白聚糖。在标记培养基中检测到一种小的、Sepharose CL - 6B Kav 0.14硫酸皮肤素 - ³⁵SO₄蛋白聚糖，它在早期（0 - 0.5小时）和晚期（6 - 24小时）追踪培养基中均有释放。它含有38 kDa的4 - 硫酸化³⁵S - GAGs，其中艾杜糖醛酸含量高，还有一个45 kDa的蛋白核心。使用双糖链蛋白聚糖或蛋白聚糖 - I抗体进行蛋白质印迹分析，在培养基中检测到了分子量相似的蛋白核心（Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571 - 4576）。在细胞层中未检测到这种³⁵S - 蛋白聚糖。然而，在细胞内隔室中存在一种几乎没有或没有蛋白核心的小硫酸皮肤素 - ³⁵SO₄。一种大的、Sepharose CL - 6B排阻的硫酸软骨素 - ³⁵SO₄蛋白聚糖释放到培养基中，并在追踪培养基的6至24小时之间被检测到。它在缔合和解离的Sepharose CL - 4B柱的空体积附近洗脱。它含有30 kDa的4 - 和6 - 硫酸化³⁵S - GAGs以及一个253 kDa的蛋白核心。在去污剂可溶和不可溶的细胞层隔室中均检测到具有相似大小³⁵S - GAGs的硫酸软骨素 - ³⁵SO₄蛋白聚糖。一种具有220 kDa蛋白核心和38 kDa³⁵S - GAGs的Sepharose CL - 6B Kav 0.11硫酸肝素 - ³⁵SO₄蛋白聚糖迅速从细胞层释放。这种蛋白聚糖比先前在分离的大鼠肾小球或肾小球基底膜中描述的要大，但核心蛋白的大小与先前在这些组织中检测到的相似。在去污剂不溶性细胞层隔室中存在一种具有更大³⁵S - GAGs的更大的硫酸乙酰肝素 - ³⁵SO₄蛋白聚糖。培养的肾小球系膜细胞释放的蛋白聚糖类似于培养的主动脉平滑肌细胞合成的或从主动脉中提取的蛋白聚糖，这支持了这些细胞起源于血管的观点。
