Yaoita E, Oguri K, Okayama E, Kawasaki K, Kobayashi S, Kihara I, Okayama M
Clinical Research Institute, National Nagoya Hospital, Japan.
J Biol Chem. 1990 Jan 5;265(1):522-31.
Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.
通过肾小球长期培养筛选出的大鼠系膜细胞在汇合状态下呈现出高低起伏的外观，并用波形蛋白和结蛋白抗体进行染色，表明它们是平滑肌样系膜细胞。细胞产生的糖缀合物用[35S]硫酸盐和[3H]葡糖胺进行代谢标记，并用含有0.5% Triton X - 100的4 M盐酸胍提取。放射性标记的糖缀合物在DEAE - Sephacel上分离，并与在相同条件下标记的肾小球合成的糖缀合物进行比较。在肾小球合成的三种主要硫酸化糖缀合物中，硫酸化糖蛋白（占总35S标记大分子的17%）、硫酸乙酰肝素蛋白聚糖（35%）和硫酸软骨素蛋白聚糖（30%），培养的系膜细胞主要合成硫酸软骨素蛋白聚糖（超过90%）。通过CsCl密度梯度离心纯化后，细胞层的硫酸软骨素蛋白聚糖在Bio - Gel A - 5m上分离为三种分子种类，估计相对分子质量（Mr）值分别为230,000、150,000和40,000 - 10,000，而释放到培养基中的由单一相对分子质量为135,000的种类组成。在β-消除反应中，前两种较大的蛋白聚糖释放出表观相对分子质量为30,000的硫酸软骨素链，而培养基中的后者释放出相对分子质量为36,000的糖胺聚糖链。细胞层中最小的蛋白聚糖在β-消除后相对分子质量没有明显变化，表明该种类如果有肽的话也只有少量。用软骨素酶AC - II和ABC分析表明，所有硫酸软骨素都是共聚物，由在己糖胺部分4位（53 - 57%）或4和6位（10 - 14%）具有硫酸基团的葡糖醛酸基 - N - 乙酰半乳糖胺（65 - 74%）和在己糖胺部分4位（17 - 23%）或4和6位（约3%）具有硫酸基团的艾杜糖醛酸基 - N - 乙酰半乳糖胺（21 - 26%）组成；即硫酸软骨素H型。培养的系膜细胞合成的硫酸软骨素H蛋白聚糖的这些特征与肾小球合成的蛋白聚糖非常相似。因此，我们得出结论，大多数（如果不是全部）肾小球硫酸软骨素蛋白聚糖是由系膜细胞合成的。还发现培养的系膜细胞合成透明质酸的水平与硫酸软骨素蛋白聚糖相似。基于这种糖胺聚糖的特征，我们讨论了系膜细胞产生的透明质酸可能的作用。
